The modulatory effects of the anxiolytic etifoxine on GABAA receptors are mediated by the β subunit
Introduction
In the mammalian brain, the inhibitory activity of type A gamma-aminobutyric acid receptors (GABAARs) can be potently enhanced by allosteric modulators such as benzodiazepines, neurosteroids or barbiturates. This property underlies the pharmacological treatment of diverse neuropsychiatric disorders like anxiety, insomnia or muscular spasms. Unfortunately, classical benzodiazepines display major side effects including anterograde amnesia, impairment of locomotor activity, ethanol-potentiation, tolerance and rebound effects on cessation of treatment (Woods and Winger, 1995). In contrast, neurosteroids exhibit less detrimental side effects but are not yet exploited on a large scale for their therapeutic potential (Gasior et al., 1999). Etifoxine (2-ethylamino-6-chloro-4-methyl-4-phenyl-4H-3,1-benzoxazine hydrochloride, Stresam®), a molecule structurally unrelated to benzodiazepines and neurosteroids, has also revealed anxiolytic properties in rodents (Boissier et al., 1972, Schlichter et al., 2000) and humans (Servant et al., 1998), without sedative, myorelaxant and mnesic side effects at anxiolytic concentrations (Micallef et al., 2001).
Recent binding and electrophysiological experiments have demonstrated that etifoxine binds to GABAARs via an allosteric site which differs from that of benzodiazepines and neurosteroids (Verleye et al., 1999, Schlichter et al., 2000, Verleye et al., 2001, Verleye et al., 2002). In addition, etifoxine increases GABAergic neurotransmission by an indirect mechanism involving the activation of peripheral (mitochondrial)-type benzodiazepine receptors and, very likely, an enhancement of neurosteroid synthesis (Schlichter et al., 2000).
Mammalian GABAARs are hetero-pentamers assembled from distinct subunit families with multiple subtypes (α1–6, β1–3, γ1–3, δ, ϵ, π and θ). The most abundant native receptors, however, are composed of only α, β and γ subunits with presumably a 2α:2β:1γ stoichiometry (Sieghart et al., 1999, Mehta and Ticku, 1999, Knight et al., 2000). The subunit composition of GABAARs has important functional implications for the effects of positive modulators like benzodiazepines and neurosteroids. Both α and γ subunits are required for the high-affinity binding of benzodiazepines, thus suggesting that the binding site is located at the α–γ interface (Sigel and Buhr, 1997). By contrast, the modulation of GABA responses by neurosteroids does not require the presence of α or γ subunits but depends on the presence of a β subunit (Mehta and Ticku, 1999). The location of the binding site of etifoxine and its mechanism of action are unknown at present. In order to address these questions, as a first step, we used a recombinant strategy to determine what types of subunits underlie its modulatory effects. For this purpose, we analysed the ability of etifoxine to modulate GABA-evoked chloride currents in Xenopus oocytes expressing one (βx), two (α1βx, βxγ2s) or three (α1βxγ2s) murine GABAAR subunits. The α1 and γ2s subunits were selected for co-expression with β subunits because they are the most abundant representatives of their subunit family in many regions of the rodent brain and, therefore, are likely to co-assemble with a β subunit to produce native GABAAR subtypes (Pirker et al., 2000). In complementary experiments, we also examined the effects of replacement of α1 by α2 and the effects of mutating β2 N-289, a residue that is known to control the interaction of several other positive modulators with GABAA receptors (Wafford et al., 1994, Belelli et al., 1997, Halliwell et al., 1999).
Section snippets
Preparation of cloned cDNAs
cDNAs encoding the murine α1, β1–3 and γ2s GABAAR subunits, subcloned into the pGW1 (=pRK5) vector downstream of the cytomegalovirus promoter, were provided by Pr. S.J. Moss (University College, London, UK). The rat α2 and β2(N289S) GABAAR cDNAs, which encode the same protein as their murine homolog, were provided by Pr. H. Lüddens (University of Mainz, Mainz, Germany) and Pr. N.L. Harrison (Cornell University, New York, USA), respectively. The identity of the wild-type and mutated β2 subunits
Homomeric receptors
In accordance with earlier reports (Connolly et al., 1996a, Connolly et al., 1996b; Krishek et al., 1996, Wooltorton et al., 1997, Cestari et al., 1996, Cestari et al., 2000, Sugimoto et al., 2000), none of the murine β subunits formed GABA-sensitive receptors when expressed alone in Xenopus oocytes. However, most β cDNA-injected cells displayed a persistent inward chloride current that could be blocked by 100 μM picrotoxinin (PTX), indicating that β subunits can oligomerize to form
Comparison with benzodiazepines, neurosteroids and barbiturates
Our results demonstrate that the β subunit plays a major role in determining the potentiating effects of etifoxine on GABAA receptors. Whereas benzodiazepines require the simultaneous presence of an α and a γ subunit for activity at nanomolar concentrations (Sigel and Buhr, 1997, Korpi et al., 2002), etifoxine remains active in the absence of either of these two subunits. Therefore, it is clear that etifoxine does not target the high-affinity binding site of benzodiazepines which is located in
Acknowledgements
This work was supported by Biocodex Laboratories (Compiègne, France). We would like to thank Pr. Stephen J. Moss (University College, London) for the α1, β1–3 and γ2s subunits, Pr. Hartmut Lüddens (University of Mainz, Germany) for the α2 subunit and Pr. Neil L. Harrison (Cornell University, New York, USA) for the β2(N289S) subunit. We would also like to thank Pr. Rémy Schlichter (University of Strasbourg, France), Pr. Bruno Lapied (University of Angers, France) for helpful discussions, and Pr.
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