Pharmacological characterization of a novel positive modulator at α4β3δ-containing extrasynaptic GABAA receptors
Introduction
The major inhibitory neurotransmitter in the central nervous system, γ-aminobutyric acid (GABA), mediates its effects via the G protein-coupled GABAB receptor, and the ionotropic, chloride and bicarbonate gating GABAA and GABAC receptors. The GABAA receptor is composed of an assembly of five subunits deriving from a heterogenous family of proteins (α1–6, β1–3, γ1–3, δ, ε, θ, π). The subunit composition of the GABAA receptor is believed to determine its localization, being either predominantly synaptic (two α, two β and one γ subunit) mediating rapid phasic transmission, or being located outside synapses (δ subunit instead of γ subunit) thought to mediate a non-desensitizing tonic current (Farrant and Nusser, 2005). Thus, a prevailing hypothesis states that extrasynaptic GABAA receptors respond to the low ambient levels of GABA outside the synaptic release sites to affect the membrane potential and neuronal excitability (Farrant and Nusser, 2005). Although this view has been challenged by recent findings of lateral diffusion of γ-containing GABAA receptors away from synapses (Bannai et al., 2009), the differential localization of γ versus δ-containing receptors has still received substantial support (Nusser et al., 1998, Wei et al., 2003). Gaboxadol (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol (THIP)) is at therapeutically relevant concentrations a functionally selective agonist at δ-containing peri- and extrasynaptic GABAA receptors (Wafford and Ebert, 2006) with little or no effect on phasic synaptic GABAA currents (Drasbek and Jensen, 2006). Whereas several positive and negative allosteric modulators have been described for the synaptic GABAA receptors, the pharmacological tools for similar modulation of extrasynaptic GABAA receptors are very sparse. Previous studies have indicated that some neurosteroids may act as selective modulators of extrasynaptic GABAA receptors, and ethanol has likewise been reported to mediate at least some of its effects via δ-containing GABAA receptors (Belelli et al., 2006, Mody et al., 2007). However, neurosteroids and ethanol interact with other transmitter systems (glutamate/NMDA), which limits their usefulness as tools for examining the physiological role of extrasynaptic GABA receptor function. Studies by Wafford et al. (2008) have recently characterized a functionally δ-selective positive allosteric modulator, and as would be predicted, this compound selectively modulates tonic currents without affecting the inhibitory spontaneous synaptic currents (IPSCs). However, no behavioural data are available on this compound, making it difficult to evaluate the in vivo physiological consequences of positively modulating the extrasynaptic GABAA receptors.
Here, we describe the in vitro and in vivo pharmacological effects of [2-Amino-4-(2,4,6-trimethylbenzylamino)-phenyl]-carbamic acid ethyl ester (AA29504), which is structurally related to the potassium channel opener retigabine. However, AA29504 differs functionally from retigabine in its ability to potentiate GABA mediated currents passed by GABAA receptors at sub-micromolar concentrations, at which no significant increase in KCNQ2/3 and −4 mediated currents are observed. Since retigabine in binding studies has previously been shown to increase the affinity of GABA to GABAA receptors (van Rijn and Willems-van, 2003), we characterized the effects of AA29504 at cloned GABAA receptors and voltage-gated K+ channels (KCNQ) expressed in oocytes. Furthermore, the effects of AA29504 on GABAA receptor mediated synaptic and tonic currents in pyramidal cells of prefrontal cortex, and in rat models of anxiety and amygdala kindling, were investigated. Our results suggest that the observed behavioural effects can be associated with AA29504-mediated dose-dependent modulation of synaptic and, in particular, extrasynaptic GABAA receptors. A part of this work was previously presented in abstract form (Jones et al., 2007).
Section snippets
Drugs and solutions
[2-Amino-4-(2,4,6-trimethylbenzylamino)-phenyl]-carbamic acid ethyl ester (AA29504), retigabine, gaboxadol (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol (THIP)) and zolpidem were synthesized at H. Lundbeck A/S. AA29504 was synthesized according to the procedure described by Von Bebenburg et al. (1979). For all in vitro experiments, AA29504 was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM and stored in aliquots at −20 °C. For the behavioural studies, AA29504 was prepared
Compound
The chemical structure of AA29504 is shown in Fig. 1. The compound was presented for the first time by Bebenburg et al. in 1979 in a publication describing the synthesis of analogues of the analgesic and antiphlogistic drug flupirtine (Von Bebenburg et al., 1979) which is, to our knowledge, the only publication describing the compound. The authors state that the antiphlogistic properties in particular are augmented by altering flupirtine to the 2,4,6-trimethyl analogue I. Although no
Discussion
In this article, we describe the pharmacological properties of AA29504, a close analogue of retigabine, which we show to be a positive modulator of GABAA receptors in oocytes with a stronger modulatory effect at α4β3δ-containing GABAA receptors compared to α1β3γ2S-containing receptors. Also, we present the ability of AA29504 to potentiate tonic GABA currents recorded from adult rat cortical pyramidal neurons induced by gaboxadol (THIP). In line with these effects we find that AA29504 acts
Conclusions
We conclude that AA29504 is a new modulator acting at both α1β3γ2S- and even stronger at α4β3δ-containing extrasynaptic GABAA receptors, which potentiates the effects of the functionally selective extrasynaptic GABA agonist gaboxadol in oocytes and slice preparations of rat cortex. The molecular mechanism of its interaction with GABA receptors is still unknown, but we believe AA29504 can be a useful tool compound for probing the role of synaptic and extrasynaptic GABA transmission in the CNS.
Acknowledgements
We would like to thank Annette Bjørn for cloning, large-scale production and purification of the GABAA and KCNQ receptor subunits DNA for the recombinant expression in oocytes. We also would like to thank Tanya Edwards B. Nugent for assisting with the behavioural experiments. Finally we would like to thank Morten Bang Nørgaard and Preben Friis Hansen for the synthesis of AA29504.
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