Review
Modulation of native and recombinant GABAA receptors by endogenous and synthetic neuroactive steroids

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Abstract

Upon administration, certain pregnane steroids produce clear behavioural effects including, anxiolysis, sedation, analgesia, anaesthesia and are anti-convulsant. This behavioural profile is characteristic of compounds that act to enhance the actions of GABA acting at the GABAA receptor. In agreement, numerous studies have now demonstrated these steroids to be potent, positive allosteric modulators of the GABAA receptor. The pregnane steroids are synthesized in the periphery by endocrine glands such as the adrenals and the ovaries, but are also made by neurons and glial cells in the central nervous system itself. Hence, these compounds could play both an endocrine and a paracrine role to influence neuronal excitability by promoting inhibition. Here we review evidence that the pregnane steroids are highly selective and extremely potent GABAA receptor modulators and that their effects at ‘physiological’ concentrations (low nanomolar) may be influenced by the subunit composition of the GABAA receptor. This feature may underlie recent findings demonstrating the effects of the neurosteroids on inhibitory synaptic transmission to be brain region dependent, although recent reports suggest that phosphorylation mechanisms may additionally influence neurosteroid sensitivity of the GABAA receptor. Numerous synthetic steroids have been synthesized in an attempt to therapeutically exploit the behavioural effects of the pregnane steroids and progress with this approach will be discussed. However, the demonstration that the steroids may be made within the central nervous system offers the alternative strategy of targeting the enzymes that synthesize/metabolise the neurosteroids to exploit this novel endocrine/paracrine interaction.

Introduction

The rapidity with which certain pregnane steroids induce sedation and anesthesia is incompatible with a genomic locus of action [82]. The demonstration in the mid-1980s that these steroids potently and selectively enhanced the actions of GABA acting at the major inhibitory receptor in the central nervous system, the GABAA receptor, provided a more feasible and logical target [39].The GABAA receptor is a member of the cysteine–cysteine loop transmitter-gated ion channel family that includes glycine, nicotinic and 5-HT3 receptors. The binding of GABA to this receptor opens an associated chloride selective ion channel that increases neuronal membrane conductance, effectively shunting the influence of excitatory neurotransmitters such as glutamate [55]. The GABAA receptor is the target for a number of therapeutically important drugs including benzodiazepines, barbiturates and general anaesthetics such as propofol and etomidate. The pregnane steroids share many of behavioural actions of these compounds being anxiolytic, anticonvulsant, sedative, analgesic and at high doses anaesthetic [32], [52]. The GABAA receptor is a heteropentamer drawn from a repertoire of α1–6, β1–3, γ1–3, δ, ε, π, θ and ρ1–3 subunits [5], [6]. Importantly, these proteins have a distinct distribution within the central nervous system and subunit composition influences not only the physiological, but additionally the pharmacological properties of the receptor. Furthermore, recent studies utilizing transgenic mice suggest that some of the behaviours produced by the benzodiazepines are mediated by distinct receptor isoforms (e.g. their sedative and amnesic actions are α1-dependent, the anxiolytic effects being α2-dependent [77]). In view of this precedent, this review will discuss in detail the influence of GABAA receptor subunit composition upon positive allosteric regulation by pregnane steroids. Although subunit dependency, as assessed from studies performed on recombinant receptors, appears to be far more subtle than that documented for the benzodiazepines, it may nonetheless be relevant in the context of concentrations of the steroids that are the likely to occur in vivo under various physiological conditions. Moreover, there are strong indications that GABA-ergic synaptic transmission may be differentially regulated by pregnane steroids in different brain regions, an effect that might be attributable to variations in GABAA receptor subunit composition.

Section snippets

The interaction of pregnane steroids with transmitter-gated ion channels

The glycine-gated chloride channel is a genetic close relation of the GABAA receptor and is composed of five transmembrane crossing subunits selected from one β and four α subunits [10]. In addition to enhancing the function of GABAA receptors certain general anaesthetics can additionally act as positive allosteric modulators of the glycine receptor [9], [10]. By contrast, endogenous pregnane steroids such as 3α-hydroxy-5α-pregnan-20-one (3α,5α-TH PROG) and the synthetic anaesthetic alphaxalone

Neurosteroid modulation of the GABAA receptor: isoform selectivity

The GABAA receptor is composed of five subunits drawn from a repertoire that includes: α1–6, β1–3, γ1–3, δ, ε, π, θ and ρ1–3 [5], [6]. Subunit composition influences both the physiological and pharmacological properties of the receptor, and, importantly, these subunits have a distinct distribution throughout the central nervous system [70], [92]. The behavioural profile of the pregnane steroids is in some respects similar to that of the benzodiazepines, e.g. having anxiolytic, anticonvulsant

Neurosteroid modulation of GABAA receptors: molecular mechanism of action

Early experiments that investigated the influence of the anesthetic pregnane steroids on GABA-evoked membrane current noise and GABA-evoked single channels demonstrated that nanomolar concentrations of these compounds had no effect on the single channel conductance of the GABAA receptor, but acted primarily to prolong the mean channel open time [4], [19], [23], [53]. Additionally, these studies revealed that at concentrations in excess of those required for enhancement of GABA-evoked responses,

Neurosteroids and inhibitory synaptic transmission

The first studies which addressed this issue used hippocampal neurones in culture and demonstrated that pregnane steroids such as alphaxalone and 3α,5α-TH PROG prolonged the decay of evoked inhibitory postsynaptic currents (IPSCs), but had little effect upon the amplitude, or the rise time of these synaptic currents [40] (Fig. 1). More recently, the pregnane steroids have been shown to produce a similar, selective prolongation of the decay of evoked IPSCs, miniature inhibitory postsynaptic

Structure activity relationships for steroids at the GABAA receptor

Studies conducted in the mid- and late-1980s exploring the structural requirements for steroid modulation of the GABAA receptor found optimal activity to be associated with structures containing a 5α- or 5β-reduced pregnane (or androstane) skeleton, a hydroxyl substituent at C3 of the steroid A ring in the α orientation and a keto group at either C20 of the pregnane steroid side chain, or C17 of an androstane ring system [33], [41], [69] (Fig. 2). The naturally occurring steroids 3α,5α-TH PROG

Multiple steroid binding sites upon the GABAA receptor

The existence of multiple GABAA receptor isoforms may provide for binding sites at which steroids display differing affinities and/or efficacies. A recent study examining the effect of synthetic enantiomers of 3α,5α-TH PROG and 3α,5β-TH PROG provides evidence for such heterogeneity in steroid action [24]. Across in vivo and in vitro assays that included loss of the righting reflex in tadpoles and mice, displacement of [35S]TBPS binding from rat brain membranes and potentiation of GABA-evoked

Concluding remarks

Neurosteroids such as 3α,5α TH PROG are clearly established as the most potent endogenous modulators of GABAA receptor-mediated inhibitory neurotransmission. The demonstration that de novo neurosteroid synthesis both in glial and neuronal cells results in levels sufficient to modulate GABAA receptor function argues in favour of a physiological/pathophysiological role for these locally released modulators. Although the pharmacological selectivity of neurosteroids versus different GABAA receptor

Acknowledgments

Some of 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’, QLK1-CT-2000-00179. Dr D. Belelli is an MRC Senior Fellow and Dr S.C. Harney was supported by an MRC Studentship. Dr B. Frenquelli held a Caledonian Research Foundation Fellowship.

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