Elsevier

Neuropharmacology

Volume 43, Issue 4, September 2002, Pages 651-661
Neuropharmacology

The influence of subunit composition on the interaction of neurosteroids with GABAA receptors

https://doi.org/10.1016/S0028-3908(02)00172-7Get rights and content

Abstract

The influence of the subunit composition of human GABAA receptors upon the GABA-modulatory properties of 5α-pregnan-3α-ol-20-one (5α,3α) has been examined using the Xenopus laevis oocyte expression system and the two electrode voltage-clamp technique. Steroid potency (EC50) is modestly influenced by the α-isoform (αxβ1γ2L; x=1–6). α2-, α4- and α5-containing receptors are significantly less sensitive to the action of low concentrations of 5α,3α (10–100 nM) when compared to α1,3,6β1γ2L receptors. Additionally, the maximal effect of the steroid is favoured at α6-containing receptors. The β-isoform (α1βyγ2L; y=1–3) has little influence on the GABA-modulatory effect of the neurosteroid. The EC50 of 5α,3α is only modestly influenced by the omission of the γ2 subunit (α1β1γ2L vs α1β1): while the maximal effect is favoured by the binary complex. However, the identity of the γ subunit influences the GABAA-modulatory potency of 5α,3α with γ2- and γ1-containing receptors being the most and the least sensitive to 5α,3α, respectively. Finally, incorporation of the ε, or δ subunit dramatically reduces and augments the GABA-enhancing actions of the steroid, respectively. These findings provide evidence that 5α,3α discriminates amongst recombinant receptors of varied subunit composition. Furthermore, this selectivity may contribute to their neuronal specificity and behavioural profile.

Introduction

GABAA receptors mediate the majority of fast inhibitory synaptic transmission in the mammalian central nervous system (CNS). A number of clinically important drugs including benzodiazepines, barbiturates and general anaesthetics such as propofol and etomidate act as positive allosteric modulators of this receptor (Barnard, 2001). Amongst the agents that enhance inhibitory transmission, certain neurosteroids (i.e. steroids synthesised in the CNS) are of particular interest. The most potent neurosteroids are active at exceptionally low concentrations (e.g. 3–30 nM for the progesterone metabolite 5α-pregnan-3α-ol-20-one (5α,3α)—see Belelli et al. (1996a)) and are both selective and enantioselective for the GABAA receptor (Lambert et al., 2001), suggesting the presence of a specific steroid binding site on the receptor protein. Consistent with enhancement of GABAA receptor function, such steroids, upon administration to rodents, exhibit clear behavioural effects including anxiolysis, analgesia, sedation, hypnosis, are potent anticonvulsants and at higher doses induce general anaesthesia (Belelli et al., 1989, Lambert et al., 1995). Attempts to exploit these behavioural properties for therapeutic advantage are ongoing (Gasior et al., 1999, Goodchild et al., 2001, Lambert et al., 2001).

It is proposed that such neurosteroids may play a physiological, or pathophysiological role to locally modulate neuronal excitability by “fine-tuning” the actions of GABA acting at the brain’s major inhibitory receptor. Support for this scenario includes: (1) concentrations of steroids achieved physiologically are sufficient to augment GABAA receptor mediated responses (Paul and Purdy, 1992, Belelli et al., 1996a, Belelli et al., 1996b); (2) the GABA-active steroids can be synthesised de novo by certain glial cells and neurones (Mellon et al., 2001); (3) administration of a 5α-reductase inhibitor causes a large decrease in brain 5α,3α levels (~90%) and concomitant changes in behaviour and GABAA receptor function, consistent with the loss of a neurosteroid inhibitory tone (Matsumoto et al., 1999, Pinna et al., 2000, Reddy and Rogawski, 2002); (4) the plasma levels of these steroids are inversely correlated with the occurrence of certain neurological and psychiatric disorders associated with inhibitory dysfunction (Rosciszewska et al., 1986, Wang et al., 1996).

Distinct subtypes of the GABAA receptor exist, resulting from the assembly of five subunits, drawn from a repertoire of 6α, 3β, 3γ, δ, ε, θ and π (Barnard, 2001). Importantly, such receptors are heterogeneously expressed in the CNS and exhibit distinctive physiological and pharmacological properties (Whiting et al., 1995, Sieghart, 2000). Furthermore, innovative experiments with so called “knock in” mice genetically engineered to express benzodiazepine-insensitive GABAA receptors suggest that different components of the behavioural repertoire of diazepam are mediated by distinct GABAA receptor isoforms (e.g. sedation and anxiolysis being mediated by α1- and α2-subunit containing receptors, respectively (Mohler et al., 2002)) and it is plausible to assume a similar scenario for the behavioural effects of the neurosteroids.

Hence, to better understand both the pharmacological and putative physiological and pathophysiological effects of the pregnane steroids it is important to establish whether their influence will be globally experienced at all GABA-ergic synapses throughout the CNS, or whether their effects will be more restricted. Although biochemical, radioligand binding and 36Cl flux studies with rat brain homogenates and synaptosomes had suggested a heterogeneity of neurosteroid/GABAA receptor recognition sites, it is only recently that electrophysiological studies have confirmed that synaptic GABAA receptors are influenced by the pregnane steroids in a neurone-dependent manner (Cooper et al., 1999, Brussaard and Herbison, 2000, Lambert et al., 2001). These observations could reflect a differential interaction of the neurosteroid with different GABAA receptor isoforms.

Although the interaction of neurosteroids with recombinant GABAA receptors has been previously investigated, the number of receptor subtypes studied has been limited. Here, we have utilised the two-point voltage-clamp technique and the Xenopus laevis oocyte expression system to determine, under identical conditions, the impact of the α1–6, β1–3, γ1–3, ε and δ subunits on the GABA-modulatory properties of 5α,3α. This study demonstrates that the effects of this neurosteroid are dependent upon the subunit composition of the GABAA receptor and that this selectivity may contribute to their neuronal specificity.

Section snippets

Preparation of in vitro transcripts, expression and electrophysiological recordings

The human GABA α1–6, β1–3, γ1–3, ε and the rat δ cDNAs were linearized in the pCDM8 and pCDNA3.1 vectors, respectively, according to standard protocols (Belelli et al., 1996b). The ε and δ subunit cDNAs were kindly provided by E. Kirkness and R.L. MacDonald, respectively, all other cDNAs were supplied by P. Whiting. Capped cRNA transcripts were prepared in vitro using T7 RNA polymerase. The integrity of the RNA transcripts was determined by denaturating gel electrophoresis.

Xenopus laevis

The influence of the α subunit on neurosteroid modulation

GABA modulation by benzodiazepines is highly dependent on the α isoform, with α4- and α6-containing receptors being insensitive to classical benzodiazepines such as diazepam (Sieghart, 2000). By contrast, the isoform of the α subunit (1–6), when co-expressed with β1 and γ2L subunits, did not greatly influence the GABA-modulatory actions of 5α,3α (Fig. 1; Table 1). Hence, irrespective of the α isoform, 5α,3α produced a concentration dependent increase of the current evoked by an EC10

Discussion

Certain pregnane steroids may act in the CNS as endogenous regulators of inhibitory neurotransmission mediated by GABAA receptors. In elucidating their putative physiological and pathophysiological roles and in assessing their therapeutic potential, it will be important to ascertain whether, given the ubiquitous expression of the GABAA receptor throughout the CNS, the effects of these compounds are universally experienced, or are neurone specific. In support of the latter, recent

Acknowledgements

The work reported here was supported by the MRC and by an EC Bioscience and Health Grant BMH4-CT97-2359 and by financial support of the Commission of the European Communities, RTD Programme ‘Quality of Life and Management of Living Resources’, QLK6-CT-2000-00179. Dr D. Belelli is an MRC Senior Fellow. The authors would like to thank Dr S. Dunn for her advice regarding expression of the δ subunit.

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    Present address: Peripheral Neuropathy Unit, Department of Neuroscience, Imperial College, Hammersmith Hospital Campus, Ducane Road, London W12 0HS, UK.

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