ReviewModulation of native and recombinant GABAA receptors by endogenous and synthetic neuroactive steroids
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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