Pharmacological Characterization of Agonists at -Containing GABAA Receptors: Functional Selectivity for Extrasynaptic Receptors

Several groups have characterized the pharmacology of 4or 6 3 -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 subunit and little on the and 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 4 3, 4 3 , and 6 3 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 4 3 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 ( 1–6, 1–3, 1–3, , , , and 1–3). Most native GABAA receptors are composed of two , two , and a , , or 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 subunits, -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 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 1 2 and 1 6 2 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 Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. 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. 0022-3565/06/3163-1351–1359$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 316, No. 3 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 92403/3078736 JPET 316:1351–1359, 2006 Printed in U.S.A. 1351 at A PE T Jornals on July 1, 2017 jpet.asjournals.org D ow nladed from 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 GABAA receptor agonist gaboxadol may be the extrasynaptically located receptors. Expression of 4 3 -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 4 3 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 1–6 x 2 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)-3isothiazolol (thio-4-PIOL). Furthermore, we have investigated the interaction of the same agonists with the cerebellar equivalent of -containing GABAA receptors, namely the receptor subtype composed of 6 3 subunits, to determine whether the altered pharmacology could be ascribed to the presence of the 4 or the subunit. Materials and Methods cRNA Preparation. Cloning and sequencing of cDNAs encoding human 4, 6, 3, and 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 ( 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 g/l streptomycin and filtered through nitrocellulose. Two-Electrode Voltage Clamping. Oocytes were placed in a 60l 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 6to 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 coapplica-