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Neurosteroid modulation of GABAA receptors: Molecular determinants and significance in health and disease
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Neurochemistry International 52 (2008) 588–595 www.elsevier.com/locate/neuint
Neurosteroid modulation of GABAA receptors: Molecular determinants and significance in health and disease Elizabeth A. Mitchell, Murray B. Herd, Benjamin G. Gunn, Jeremy J. Lambert, Delia Belelli * Neurosciences Institute, Division of Pathology and Neuroscience, University of Dundee, Ninewells Hospital and Medical School, Ninewells Hospital, Dundee DD1 9SY, United Kingdom Received 1 August 2007; received in revised form 30 September 2007; accepted 10 October 2007 Available online 16 October 2007
Abstract Over the past 20 years it has become apparent that certain steroids, synthesised de novo in the brain, hence named neurosteroids, produce immediate changes (within seconds) in neuronal excitability, a time scale that precludes a genomic locus of action. Identified molecular targets underlying modulation of brain excitability include both the inhibitory GABAA and the excitatory NMDA receptor. Of particular interest is the interaction of certain neurosteroids with the GABAA receptor, the major inhibitory receptor in mammalian brain. During the last decade, compelling evidence has accrued to reveal that locally produced neurosteroids may selectively ‘‘fine tune’’ neuronal inhibition. A range of molecular mechanisms including the subunit composition of the receptor(s), phosphorylation and local steroid metabolism, underpin the regionand neuronal selectivity of action of neurosteroids at synaptic and extrasynaptic GABAA receptors. The relative contribution played by each of these mechanisms in a variety of physiological and pathophysiological scenarios is currently being scrutinised at a cellular and molecular level. However, it is not known how such mechanisms may act in concert to influence behavioural profiles in health and disease. An important question concerns the identification of the anatomical substrates mediating the repertoire of behaviours produced by neurosteroids. ‘‘Knock-in’’ mice expressing mutant GABAA subunits engineered to be insensitive to benzodiazepines or general anaesthetics have proved invaluable in evaluating the role of GABAA receptor subtypes in complex behaviours such as sedation, cognition and anxiety [Rudolph, U., Mohler, H., 2006. GABA-based therapeutic approaches: GABAA receptor subtype functions. Curr. Opin. Pharmacol. 6, 18–23]. However, the development of a similar approach for neurosteroids has been hampered by the limited knowledge that, until recently, has surrounded the identity of the amino acid residues contributing to the neurosteroid binding pocket. Here, we will review recent progress in identifying the neurosteroid binding site on the GABAA receptor, and discuss how these discoveries will impact on our understanding of the role of neurosteroids in health and disease. # 2007 Elsevier Ltd. All rights reserved. Keywords: Neurosteroid binding site; GABAA receptor delta subunit; Extrasynaptic conductance; Anxiolytic; ‘‘Knock-in’’ mice
1. Introduction The neurotransmitter GABA, acting through GABAA receptors, mediates the majority of ‘‘fast’’ inhibitory synaptic signalling in the mammalian central nervous system (CNS) and consequently has a profound influence on our mood and behaviour. Additionally, it has recently been recognized that GABAA receptors underpin a sustained, tonic form of inhibition via extrasynaptically located receptors, which play a significant
* Corresponding author. Tel.: +44 1382 632161; fax: +44 1382 667120. E-mail address: [email protected] (D. Belelli). 0197-0186/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2007.10.007
role in brain excitability, both in normal and pathophysiological states (Semyanov et al., 2004; Farrant and Nusser, 2005). GABAA receptors are an important target for a variety of clinically important drugs including the benzodiazepines (used in the treatment of anxiety, epilepsy, sleep disorders and alcohol withdrawal, etc.) and a number of general anaesthetics such as propofol, etomidate and thiopental that are used to induce anaesthesia (Sieghart, 1995; Sigel and Buhr, 1997; Belelli et al., 1999; Rudolph and Mohler, 2006). These compounds all act as positive allosteric modulators to potentiate inhibition mediated by GABAA receptors. A variety of neurological and psychiatric disorders, which include epilepsy, pain syndromes, anxiety, depression and schizophrenia exhibit perturbations of GABAergic transmission. Interestingly, some of these conditions have
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been associated with abnormal levels of certain endogenously occurring neurosteroids, which potently and selectively enhance the function of the GABAA receptor via a distinct site (reviewed in Belelli and Lambert, 2005; Akk et al., 2007; Herd et al., 2007; Hosie et al., 2007). Neurosteroids, typified by the potent progesterone metabolite, 5a-pregnan-3a-ol-20-one (3a,5a-THPROG) can be synthesised de novo in the CNS, both in neurons and glia in levels sufficient to modulate GABAA receptor function and thus, are likely to play an important physiological/pathophysiological role (Belelli and Lambert, 2005; Herd et al., 2007). Indeed, a direct link between abnormal neurosteroid synthesis and brain function has been documented together with the demonstration of sex differences in the genomic regulation of the enzymes responsible for their synthesis and degradation (Matsui et al., 2002; Mitev et al., 2003; Reddy, 2003; Torres and Ortega, 2003; Pinna et al., 2004). Furthermore, concomitant with the demonstration of the trophic actions of GABA in the developing CNS (e.g. in neuronal proliferation, differentiation, migration and synapse establishment; Represa and Ben Ari, 2005), a role for neurosteroids in neurodevelopment has begun to emerge (Grobin et al., 2006). Although a persuasive body of indirect evidence has accumulated over the past 20 years to support the existence of a specific neurosteroid binding site(s) on the receptor protein, attempts to identify such a site(s) have proved, until lately, unsuccessful. Recently, the application of molecular modelling of chimaeric constructs made of steroid-sensitive and insensitive GABA subunits has identified putative neurosteroid binding sites and revealed specific amino acid residues that govern the steroid/receptor interaction. The molecular mechanisms underpinning the selectivity of steroid action at synaptic and extrasynaptic GABAA receptors have been reviewed recently (Herd et al., 2007). Here, we specifically consider advances in defining putative neurosteroid binding pockets on the GABAA receptor protein. We then speculate on how this new information may be exploited to advance our understanding of the role of neurosteroids in health and disease. 2. GABAA receptor heterogeneity Advances in our understanding of neurosteroids are in part intrinsically linked to progress in elucidating the complex roles different GABAA receptor isoforms play in neuronal signalling, development and behaviour. The GABAA receptor is a member of the Cys-loop family of transmitter-gated ion channels and is composed of five trans-membrane subunits arranged to form an intrinsic anion-conducting channel. The cloning of the first subunits (a1 and b1) in the late 1980s led to the subsequent discovery of a further 17 subunits (a1–6, b1–3, g1–3, d, e, u, p and r1–3) classed according to sequence homology (70% within a subunit class, 30% between classes), a heterogeneity further increased by alternative exon splicing (Barnard et al., 1998; Sieghart, 2006). In the absence of rules governing subunit co-assembly, this repertoire could theoretically underpin the existence of thousands of distinct
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GABAA receptor subtypes. However, strict subunit assembly, trafficking requirements and region-selective subunit expression patterns (Sieghart and Sperk, 2002; Fritschy and Brunig, 2003; Luscher and Keller, 2004), refine this number to 30 main GABAA receptor subtypes, each with distinct physiological and pharmacological properties. The majority of GABAA receptors in vivo are composed of a, b, and g subunits, in a probable stoichiometry of 2a, 2b to 1g subunit. By far the most abundant subtype is the a1b2g2 isoform (60%) with a2b3g2 and a3bxg2 representing 15–20% and 10–15% of the total, respectively (Mohler, 2007). Receptors incorporating a4, a5, a6, b1, g1, g3, d, e, and u subunits are less numerous, but nevertheless serve important functions. The d and e subunits appear to substitute for the g subunit and receptor isoforms incorporating the d subunit specifically localise to extrasynaptic/perisynaptic regions in selective neuronal populations (e.g. cerebellar and dentate gyrus granule cells, thalamic relay nuclei and neocortex; Nusser et al., 1996, 1998; Brickley et al., 2001; Nusser and Mody, 2002; Stell et al., 2003; Belelli et al., 2005; Cope et al., 2005; Chandra et al., 2006; Drasbek and Jensen, 2006; Bright et al., 2007). The r subunits form both homomeric and heteromeric GABA-gated chloride channels with distinct pharmacological properties leading some researchers to separately characterise these receptors as GABAC. However, evidence that, in vivo, these subunits may co-assemble with members of the GABAA receptor subunit class may warrant, in the near future, their inclusion in the GABAA receptor category (Milligan et al., 2004; Harvey et al., 2006). The subunit composition influences fundamental features of the receptor including the sensitivity to GABA, channel kinetics, desensitisation, pharmacological properties and the neuronal location (e.g. synaptic/extrasynaptic). Thus, as noted above, receptors incorporating the d subunit, in combination with a b and, usually, either an a4, a6 or a1 subunit, are insensitive to modulation by benzodiazepines and localise at extrasynaptic sites, where they mediate a persistent form of tonic inhibition. The properties of d-containing GABAA receptors are tailored for such a role as they exhibit a relatively high affinity for GABA and limited desensitisation, features that permit a maintained response to ambient GABA (Saxena and Macdonald, 1994; Haas and Macdonald, 1999; Bianchi et al., 2002; Lagrange et al., 2007). By contrast, synaptically located receptors typically incorporate the g2 subunit in combination with different a and b subunit isoforms. In agreement, the g2 subunit is crucial for receptor clustering and synaptic localisation (Essrich et al., 1998; Fritschy and Brunig, 2003; Schweizer et al., 2003). However, g2-containing receptors may additionally localise at extrasynaptic locations (e.g. a3b2/3g2 or a5b3g2; see Devor et al., 2001; Caraiscos et al., 2004). Indeed, g2-containing synaptic GABAA receptors appear to be directly recruited, by lateral diffusion, from a dynamic pool of extrasynaptic GABAA receptors (Thomas et al., 2005; Bogdanov et al., 2006). The pharmacological and behavioural relevance of GABAA receptor subtypes has been elegantly illustrated by the
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behavioural analysis of transgenic mouse models (i.e. ‘‘knockin’’ mice). Such models were developed subsequent to the identification of specific residues within a and b subunits that govern the actions of benzodiazepines and the general anaesthetic etomidate, respectively. This approach has enabled a genetic dissection of the behavioural repertoire associated with the systemic administration of such agents. Thus, for example, the sedative, amnestic and a component of the anticonvulsant effects of diazepam are mediated by receptors incorporating the a1 subunit, whereas the anxiolytic and myorelaxant actions of this benzodiazepine implicate a role for a2-GABAA receptors with a contribution from a3 and a5 subunit containing receptors, respectively (van Rijnsoever et al., 2004; Mohler, 2007). A similar approach has characterised the contribution of GABAA receptor subtypes incorporating different b subunit isoforms to the variety of behaviours associated with general anaesthetics, e.g. the sedative and immobilising actions of etomidate are mediated by b2- and b3-containing GABAA receptors, respectively (Jurd et al., 2003; Reynolds et al., 2003). Whether similar receptor isoforms segregate with the behavioural repertoire associated with neurosteroids remains to be determined. In this context, of specific interest is the role of the benzodiazepine-insensitive dcontaining extrasynaptic GABAA receptors, which have been proposed to represent a significant molecular target underpinning many of the physiologically relevant actions of neurosteroids (e.g. anxiolytic and anticonvulsant effects; Maguire et al., 2005, see below). 3. A distinct neurosteroid binding site 3.1. Indirect evidence Although the GABAA receptor-active neurosteroids such as 3a,5a-THPROG, are highly lipid soluble (oil to water coefficient > 10,000), their potent and stereoselective interaction with native GABAA receptors suggested a specific neurosteroid recognition site on the receptor protein (Callachan et al., 1987; Harrison et al., 1987; Peters et al., 1988). This concept was bolstered by the subsequent observation that GABAA receptor-active steroids exhibit a clear enantioselectivity, both in vitro and in vivo (Wittmer et al., 1996), a finding indicative of a selective interaction within a chiral environment, traditionally afforded by protein structures. In agreement, the effects of two pairs of anaesthetic steroid enantiomers upon lipid bilayers lack enantiomeric specificity (Alakoskela et al., 2007). Early radioligand binding and electrophysiological studies provided unambiguous evidence that the recognition site for neurosteroids was distinct from other known allosteric binding sites (e.g. benzodiazepine and barbiturates) on the GABAA receptor (Callachan et al., 1987; Gee et al., 1988; Peters et al., 1988). The discovery that neurosteroids could enhance GABAevoked responses at low nanomolar concentrations and directly activate the receptor in the submicromolar range, postulated the existence of at least two distinct binding sites (Callachan et al., 1987). However, until recently, the identification of such sites has remained elusive.
Traditional approaches aimed at identifying binding sites on receptor proteins have typically relied on the use of photolabelling agents and/or functional analysis of chimaeric constructs between ligand-sensitive and -insensitive subunits. Such methods have revealed residues that contribute to the binding pocket for benzodiazepines (Pritchett et al., 1989; Pritchett and Seeburg, 1990; Duncalfe et al., 1996; reviewed in Sigel and Buhr, 1997). Despite the potency inferred by functional studies, neurosteroid lipophilicity has hampered the development of suitable radioligands. The pregnane neurosteroids are highly selective for the GABAA receptor. However, in contrast to the exquisite subunit selectivity exhibited by benzodiazepines, recombinant expression studies have revealed a promiscuous interaction of neurosteroids with many GABAA receptor isoforms (Belelli et al., 2002). This lack of specificity has confounded the use of chimaeric constructs and mutagenesis (Rick et al., 1998; Akk et al., 2004; Ueno et al., 2004, see below). Recently, an investigation into how neurosteroids access the receptor has been pursued as an indirect approach to gain an insight into the location of the neurosteroid binding site(s) (Akk et al., 2005). Utilising the cell-attached configuration of the patch clamp technique, Akk and colleagues demonstrated that aqueous extracellular access is not a prerequisite for neurosteroid modulation of the GABAA receptor. Specifically, they revealed that the enhancement of GABA-gated channel activity produced when 3a,5a-THPROG is included in the patch pipette is indistinguishable from that caused by the bathapplication of the steroid. Furthermore, tracking the steroid access route with a fluorescent derivative of 3a,5a-THPROG, revealed prominent steroid accumulation, both in the plasma membrane and intracellularly. These findings are consistent with a route of access through the membrane, a conclusion strengthened by the observation that a membrane-impermeant steroid analogue enhances channel activity only when applied to the inner, but not to the outer, membrane leaflet (Akk et al., 2005). Collectively, these recent observations support earlier proposals (Lambert et al., 1990) that the interaction of neurosteroids with GABAA receptors may be mediated via a membrane-embedded hydrophobic pocket of the receptor protein, presumably accessed by the steroid via lateral diffusion. 3.2. Identification of the neurosteroid binding pocket As noted above, the limited selectivity exhibited by neurosteroids for different GABAA receptor isoforms has complicated identification of their binding site(s). However, the closely related glycine receptor is insensitive to such steroids, including the synthetic anaesthetic steroid alphaxalone (Harrison et al., 1987; Weir et al., 2004). Functional analysis of chimaeras composed of alphaxalone-sensitive GABAA receptor a2 or b1 subunits and the alphaxalone-insensitive glycine receptor a1 subunit, suggested a binding domain located in a region N-terminal to the TM2 domain of the a2 and/ or b1 subunit (Rick et al., 1998). Subsequent attempts employed chimaeric constructs between the glycine a1 (Ueno
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et al., 2004), or GABA r subunit (Akk et al., 2004) and the GABAA receptor a1 or g2 subunit, respectively. However, functional analysis of these chimaeras implicated a different (TM4) trans-membrane portion of the receptor protein, from that suggested by Rick et al. (1998). Nonetheless, collectively, such reports highlighted the importance of the trans-membrane regions of the protein for neurosteroid action, but did not unambiguously pinpoint their location, nor did these studies identify the residues forming the binding pocket. We and others had previously demonstrated that a GABAgated chloride channel derived from Drosophila (RDL) was relatively insensitive to neurosteroids (Chen et al., 1994; Belelli et al., 1996; Hosie and Sattelle, 1996). Recently, chimaeric constructs of RDL and mammalian GABAA subunits were used to generate neurosteroid-insensitive GABAA receptors (Hosie et al., 2006). Incorporation of the TM1 to TM2 region of the RDL GABA subunit into the murine a1, but not the b2 subunit, proved sufficient to confer insensitivity to the GABAenhancing and ‘‘agonist’’ actions of 3a,5a-THPROG and 3a,5a-tetrahydrodeoxycorticosterone (3a,5a-THDOC) determined with a heterologously expressed ternary aRDL-b-g receptor complex (Hosie et al., 2006). This advance, coupled with a retrospective re-evaluation of the steroid structure activity requirements for modulation of GABAA receptor function has proved invaluable to identifying specific amino acid residues that may contribute to the neurosteroid binding pocket. The critical importance for GABAA receptor activity of a hydroxyl and a ketone group in the C3a and C20 positions, respectively (Gee et al., 1987, 1988; Harrison et al., 1987;
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Peters et al., 1988; Lambert et al., 2001), suggested that neurosteroids would specifically interact with suitably spaced hydrophilic residues within the receptor protein. In addition, early models of the neurosteroid pharmacophore had postulated the hydrophobic nature of the four rings of the steroid backbone to be sufficient to allow access to a hydrophobic pocket within the protein (Upasani et al., 1997). The hydrogen bonding acceptor and donor properties afforded by the 3a-hydroxyl and 20-ketone groups, respectively, would provide the primary ‘‘docking points’’ necessary to confer a high degree of potency, specificity and efficacy to the neurosteroid interaction with the GABAA receptor (Upasani et al., 1997). The search for the polar, or charged residues that would allow the formation of such hydrogen bonds within the TM1–TM2 domains of the mouse a1 subunit resulted in the specific identification of threonine 236 and glutamine 241. These residues are crucial determinants of the ‘‘agonist’’ and the GABA-enhancing actions, respectively of both 3a,5a-THPROG and 3a,5aTHDOC. 3.3. Steroid-sensitive and -insensitive GABA subunits Consistent with the putative role played by residues threonine 236 and glutamine 241 in the formation of hydrogen bonds with GABAA receptor-active steroid molecules, hydrophobic/non-polar residues occupy equivalent positions in steroid-insensitive subunits (Fig. 1). Thus, a non-polar isoleucine, or methionine residue occupies the homologous position to threonine 236 in the equivalent position of the glycine a1/b and GABA r subunit, respectively. Receptors
Fig. 1. Sequence alignment of trans-membrane region (TM1) of the Drosophila RDL, human (h) GABAA, rat (r) and human glycine receptor subunits. Amino acid residues important for the GABA-enhancing (residue 241) and agonist (residue 236) actions of 3a,5a-THPROG and 3a,5a-THDOC at GABAA receptors are coloured grey for RDL, GABAA and glycine receptor subunits.
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Fig. 2. Sequence alignment of the COOH-terminal region including trans-membrane region (TM4), of the Drosophila RDL, human (h) GABAA, human and rat (r) glycine receptor subunits. Amino acid residues (407 and 410), which additionally contribute to the binding pocket mediating the GABA-enhancing actions of 3a,5aTHPROG and 3a,5a-THDOC at GABAA receptors, are coloured grey for RDL, GABAA and glycine receptor subunits.
formed from these subunits are insensitive to the actions of 5areduced steroids (Belelli et al., 1999; Weir et al., 2004; Morris and Amin, 2004). Similarly, while glutamine 241 is conserved across all the a subunit isoforms (Fig. 1), replacement of this residue by tryptophan (the residue that occupies the equivalent position of the RDL subunit) abolishes enhancement of the GABA-evoked response by the steroid (Hosie et al., 2006), probably because this aromatic residue can only act to provide hydrogen bonds. Interestingly, a tryptophan residue also occupies the homologous position within the steroid-insensitive glycine a1/b and GABA r subunits (Fig. 2). Clearly, it would be of interest to determine whether replacement of this tryptophan residue with glutamine is sufficient to impart steroid sensitivity to either the glycine a1/b, or GABA r subunits. As the C20 ketone group can only act as a hydrogen bond acceptor, collectively, these findings indicate the glutamine residue to be a crucial determinant, allowing a hydrogen bond-mediated interaction of the a subunit with the 3a hydroxyl group. Subsequent molecular modelling of the binding site, utilising the calculated distance between the two putative docking centres of the steroid molecule as a guide (i.e. the C3 hydroxyl and the C20 ketone groups), has uncovered an additional contribution made by two residues located in the TM4 domain (N407 and Y410). These amino acids appear to contribute to the proposed pocket that mediates the GABA-modulatory actions of the neurosteroids. Interestingly, the residues are conserved across the steroidsensitive and -insensitive subunits (see Fig. 2). Collectively, these findings are consistent with a contribution by the three residues to a single potentiating neurosteroid site. However, the recent demonstration that the potentiating actions of two steroids (5b-3a-hydroxy-18-norandrostane17b-carbonitrile, and the unnatural enantiomer of etiocholanolone) are not similarly influenced by mutations of these residues complicates the interpretation of the above findings
and raises the prospect of multiple steroid recognition sites (Li et al., 2006, 2007). 3.4. Implications for the putative physiological/ pathophysiological role of neurosteroids The molecular identification of the residues contributing to the sites mediating the modulatory and agonist actions of neurosteroids has finally provided the information required to allow the creation of a generation of subunit specific neurosteroid-insensitive ‘‘knock-in’’ mice. Such mice should permit a novel insight into the proposed physiological and pathophysiological roles of neurosteroids and identify which GABAA receptors mediate these steroid-induced behaviours. However, the quest to identify the neuronal substrates of neurosteroid actions in health and disease poses several challenges. Importantly, the choice of a suitable amino acid substitution, which, while conferring insensitivity to the action of neurosteroids such as 3a,5a-THPROG and 3a,5a-THDOC, should produce little or no change to the receptor sensitivity to the neurotransmitter GABA. Indeed, mice harbouring mutations that not only modify drug binding properties, but additionally alter the receptor sensitivity to GABA, may exhibit behavioural abnormalities that complicate interpretation (Homanics et al., 2005). Importantly, the glutamine to leucine 241 mutation of the a subunit abolished neurosteroid enhancement of the GABA response, but in contrast to the glutamine to tryptophan mutation, the modification additionally had a modest effect on the interaction of GABA with the receptor (Hosie et al., 2006). Although such mutations appear to have only a modest influence on recombinant GABAA receptor function, their impact on the expression, trafficking and function of native neuronal GABAA receptors will need to be verified in the appropriate ‘‘knock-in’’ mice.
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Clearly, inherent in the hypothesis that neurosteroids may physiologically influence GABAA receptor-mediated inhibition is the prediction of a behavioural phenotype, which presumably will be dependent on the a subunit isoform that is ‘‘steroidinactivated’’. To unequivocally elucidate the role played by neurosteroids in specific brain functions may require a reversible and temporally controlled method to ‘‘silence’’ the steroid sensitivity of a specific receptor isoform in a defined neuronal population (Wulff et al., 2007). Such an approach would offer the obvious advantage of overcoming the complications (e.g. compensations) arising from irreversible genetic alterations (Brickley et al., 2001) and additionally may permit the putative role of neurosteroids in neurodevelopment to be explored more meaningfully. Although the mammalian CNS expresses a palette of 20–30 major GABAA receptor subtypes, physiologically a more restricted number of receptors may be influenced by endogenous neurosteroid levels. As the ‘‘silencing’’ mutation would be introduced into the a subunit, the question arises as to which receptor populations to target. Extrasynaptic d-containing GABAA receptors, particularly those incorporating the a4 subunit, may mediate many of the behavioural actions of neurosteroids that occur for example during the ovarian cycle, stress and puberty (Maguire et al., 2005; Maguire and Mody, 2007; Shen et al., 2007). Recombinant expression studies unanimously support a preferential interaction of neurosteroids with d-containing receptor isoforms (Belelli et al., 2002; Brown et al., 2002; Wohlfarth et al., 2002), although investigations of the in vitro actions of neurosteroids such as 3a,5a-THPROG and 3a,5aTHDOC in a native environment have produced apparently inconsistent findings. Indeed, not all native d-containing extrasynaptic GABAA receptors are sensitive to low, physiological concentrations of neurosteroids (e.g. thalamic tonic conductance, Porcello et al., 2003, reviewed by Herd et al., 2007). Therefore, should a loss of steroid sensitivity be demonstrated in recombinant a4Q241Wbd GABAA receptors, investigation of steroid action in neurones putatively expressing this receptor isoform could provide an interesting ‘‘proof of concept’’ study. Ultimately, this approach could be further refined to restrict the expression of the mutant subunit to a defined neuronal population (e.g. dentate gyrus granule cells, McHugh et al., 2007; Wulff et al., 2007). Such models would permit the contribution of specific neuronal receptor populations to complex neurosteroid-induced behaviours to be dissected. However, extrasynaptic GABAA receptors may not be the only significant molecular target of neurosteroid actions. Indeed, current evidence suggests that modulation of synaptic GABAA receptors may play an equally important role. Thus, for example, synaptic a3b3g2 GABAA receptors in the spinal cord may mediate the putative endogenous analgesic actions of neurosteroids (Poisbeau et al., 2005). Clearly, the creation of mice rendered steroid-insensitive at the a3-containing GABAA receptors in neuronal populations associated with the processing of nociceptive signals (e.g. spinal cord) may be particularly instructive in defining the putative role played by neurosteroids as analgesic agents.
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4. Conclusions Neurosteroids, typified by 3a,5a-THPROG and 3a,5aTHDOC are now recognized as highly selective and potent endogenous modulators of GABAA receptor-mediated synaptic and extrasynaptic neurotransmission. The recent identification of specific residues that contribute to the neurosteroid recognition site(s) is an essential precursor to the generation of mice that express steroid-insensitive GABAA receptor isoforms. Such models should permit the physiological and pathophysiological effects of neurosteroids to be dissected in a manner analogous to benzodiazepines and general anaesthetics. The selective interaction of neurosteroids with neuronal GABAA receptors is influenced by the subunit composition of the receptor, local metabolism and phosphorylation. Therefore, this approach may provide a unique opportunity to investigate how these different mechanisms act in concert to influence neurosteroid regulation of brain excitability in health and disease. Acknowledgements Some aspects of the work described here were supported by the MRC UK, a BBSRC Project Grant and Tenovus Scotland. We thank Dr. Michelle Cooper for providing the sequence alignments. References Akk, G., Bracamontes, J.R., Covey, D.F., Evers, A., Dao, T., Steinbach, J.H., 2004. Neuroactive steroids have multiple actions to potentiate GABAA receptors. J. Physiol. 558, 59–74. Akk, G., Shu, H.J., Wang, C., Steinbach, J.H., Zorumski, C.F., Covey, D.F., Mennerick, S., 2005. Neurosteroid access to the GABAA receptor. J. Neurosci. 25, 11605–11613. Akk, G., Covey, D.F., Evers, A.S., Steinbach, J.H., Zorumski, C.F., Mennerick, S., 2007. Mechanisms of neurosteroid interactions with GABAA receptors. Pharmacol. Ther. 116, 35–37. Alakoskela, J.M., Covey, D.F., Kinnunen, P.K., 2007. Lack of enantiomeric specificity in the effects of anesthetic steroids on lipid bilayers. Biochim. Biophys. Acta 1768, 131–145. Barnard, E.A., Skolnick, P., Olsen, R.W., Mohler, H., Sieghart, W., Biggio, G., Braestrup, C., Bateson, A.N., Langer, S.Z., 1998. International Union of Pharmacology. XV. Subtypes of gamma-aminobutyric acidA receptors: classification on the basis of subunit structure and receptor function. Pharmacol. Rev. 50, 291–313. Belelli, D., Lambert, J.J., 2005. Neurosteroids: endogenous regulators of the GABAA receptor. Nat. Rev. Neurosci. 6, 565–575. Belelli, D., Callachan, H., Hill-Venning, C., Peters, J.A., Lambert, J.J., 1996. Interaction of positive allosteric modulators with human and Drosophila recombinant GABA receptors expressed in Xenopus laevis oocytes. Br. J. Pharmacol. 118, 563–576. Belelli, D., Pistis, M., Peters, J.A., Lambert, J.J., 1999. General anaesthetic action at transmitter-gated inhibitory amino acid receptors. Trends Pharmacol. Sci. 20, 496–502. Belelli, D., Casula, A., Ling, A., Lambert, J.J., 2002. The influence of subunit composition on the interaction of neurosteroids with GABAA receptors. Neuropharmacology 43, 651–661. Belelli, D., Peden, D.R., Rosahl, T.W., Wafford, K.A., Lambert, J.J., 2005. Extrasynaptic GABAA receptors of thalamocortical neurons: a molecular target for hypnotics. J. Neurosci. 25, 11513–11520. Bianchi, M.T., Haas, K.F., Macdonald, R.L., 2002. a1 and a6 subunits specify distinct desensitization, deactivation and neurosteroid modulation of
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GABAA receptors containing the d subunit. Neuropharmacology 43, 492–502. Bogdanov, Y., Michels, G., Armstrong-Gold, C., Haydon, P.G., Lindstrom, J., Pangalos, M., Moss, S.J., 2006. Synaptic GABAA receptors are directly recruited from their extrasynaptic counterparts. EMBO J. 25, 4381–4389. Brickley, S.G., Revilla, V., Cull-Candy, S.G., Wisden, W., Farrant, M., 2001. Adaptive regulation of neuronal excitability by a voltage-independent potassium conductance. Nature 409, 88–92. Bright, D.P., Aller, M.I., Brickley, S.G., 2007. Synaptic release generates a tonic GABAA receptor-mediated conductance that modulates burst precision in thalamic relay neurons. J. Neurosci. 27, 2560–2569. Brown, N., Kerby, J., Bonnert, T.P., Whiting, P.J., Wafford, K.A., 2002. Pharmacological characterization of a novel cell line expressing human a4b3d GABAA receptors. Br. J. Pharmacol. 136, 965–974. Callachan, H., Cottrell, G.A., Hather, N.Y., Lambert, J.J., Nooney, J.M., Peters, J.A., 1987. Modulation of the GABAA receptor by progesterone metabolites. Proc. R. Soc. Lond. B: Biol. Sci. 231, 359–369. Caraiscos, V.B., Elliott, E.M., You, T., Cheng, V.Y., Belelli, D., Newell, J.G., Jackson, M.F., Lambert, J.J., Rosahl, T.W., Wafford, K.A., MacDonald, J.F., Orser, B.A., 2004. Tonic inhibition in mouse hippocampal CA1 pyramidal neurons is mediated by a5 subunit-containing g-aminobutyric acid type A receptors. Proc. Natl. Acad. Sci. U.S.A. 101, 3662–3667. Chandra, D., Jia, F., Liang, J., Peng, Z., Suryanarayanan, A., Werner, D.F., Spigelman, I., Houser, C.R., Olsen, R.W., Harrison, N.L., Homanics, G.E., 2006. GABAA receptor a4 subunits mediate extrasynaptic inhibition in thalamus and dentate gyrus and the action of gaboxadol. Proc. Natl. Acad. Sci. U.S.A. 103, 15230–15235. Chen, R., Belelli, D., Lambert, J.J., Peters, J.A., Reyes, A., Lan, N.C., 1994. Cloning and functional expression of a Drosophila g-aminobutyric acid receptor. Proc. Natl. Acad. Sci. U.S.A. 91, 6069–6073. Cope, D.W., Hughes, S.W., Crunelli, V., 2005. GABAA receptor-mediated tonic inhibition in thalamic neurons. J. Neurosci. 25, 11553–11563. Devor, A., Fritschy, J.M., Yarom, Y., 2001. Spatial distribution and subunit composition of GABAA receptors in the inferior olivary nucleus. J. Neurophysiol. 85, 1686–1696. Drasbek, K.R., Jensen, K., 2006. THIP, a hypnotic and antinociceptive drug, enhances an extrasynaptic GABAA receptor-mediated conductance in mouse neocortex. Cereb. Cortex 16, 1134–1141. Duncalfe, L.L., Carpenter, M.R., Smillie, L.B., Martin, I.L., Dunn, S.M., 1996. The major site of photoaffinity labeling of the g-aminobutyric acid type A receptor by [3H]flunitrazepam is histidine 102 of the a subunit. J. Biol. Chem. 271, 9209–9214. Essrich, C., Lorez, M., Benson, J.A., Fritschy, J.M., Luscher, B., 1998. Postsynaptic clustering of major GABAA receptor subtypes requires the gamma 2 subunit and gephyrin. Nat. Neurosci. 1, 563–571. Farrant, M., Nusser, Z., 2005. Variations on an inhibitory theme: phasic and tonic activation of GABAA receptors. Nat. Rev. Neurosci. 6, 215–229. Fritschy, J.M., Brunig, I., 2003. Formation and plasticity of GABA-ergic synapses: physiological mechanisms and pathophysiological implications. Pharmacol. Ther. 98, 299–323. Gee, K.W., Chang, W.C., Brinton, R.E., McEwen, B.S., 1987. GABA-dependent modulation of the Cl-ionophore by steroids in rat brain. Eur. J. Pharmacol. 136, 419–423. Gee, K.W., Bolger, M.B., Brinton, R.E., Coirini, H., McEwen, B.S., 1988. Steroid modulation of the chloride ionophore in rat brain: structure-activity requirements, regional dependence and mechanism of action. J. Pharmacol. Exp. Ther. 246, 803–812. Grobin, A.C., Gizerian, S., Lieberman, J.A., Morrow, A.L., 2006. Perinatal allopregnanolone influences prefrontal cortex structure, connectivity and behavior in adult rats. Neuroscience 138, 809–819. Haas, K.F., Macdonald, R.L., 1999. GABAA receptor subunit g2 and d subtypes confer unique kinetic properties on recombinant GABAA receptor currents in mouse fibroblasts. J. Physiol. 514 (Pt 1), 27–45. Harrison, N.L., Majewska, M.D., Harrington, J.W., Barker, J.L., 1987. Structure-activity relationships for steroid interaction with the g-aminobutyric acidA receptor complex. J. Pharmacol. Exp. Ther. 241, 346–353.
Harvey, V.L., Duguid, I.C., Krasel, C., Stephens, G.J., 2006. Evidence that GABA r subunits contribute to functional ionotropic GABA receptors in mouse cerebellar Purkinje cells. J. Physiol. 577, 127–139. Herd, M.B., Belelli, D., Lambert, J.J., 2007. Neurosteroid modulation of synaptic and extrasynaptic GABAA receptors. Pharmacol. Ther. 116, 20–34. Homanics, G.E., Elsen, F.P., Ying, S.W., Jenkins, A., Ferguson, C., Sloat, B., Yuditskaya, S., Goldstein, P.A., Kralic, J.E., Morrow, A.L., Harrison, N.L., 2005. A gain-of-function mutation in the GABA receptor produces synaptic and behavioral abnormalities in the mouse. Genes Brain Behav. 4, 10–19. Hosie, A.M., Sattelle, D.B., 1996. Allosteric modulation of an expressed homooligomeric GABA-gated chloride channel of Drosophila melanogaster. Br. J. Pharmacol. 117, 1229–1237. Hosie, A.M., Wilkins, M.E., da Silva, H.M., Smart, T.G., 2006. Endogenous neurosteroids regulate GABAA receptors through two discrete transmembrane sites. Nature 444, 486–489. Hosie, A.M., Wilkins, M.E., Smart, T.G., 2007. Neurosteroid binding sites on GABAA receptors. Pharmacol. Ther. 116, 7–19. Jurd, R., Arras, M., Lambert, S., Drexler, B., Siegwart, R., Crestani, F., Zaugg, M., Vogt, K.E., Ledermann, B., Antkowiak, B., Rudolph, U., 2003. General anesthetic actions in vivo strongly attenuated by a point mutation in the GABAA receptor b3 subunit. FASEB J. 17, 250–252. Lagrange, A.H., Botzolakis, E.J., Macdonald, R.L., 2007. Enhanced macroscopic desensitization shapes the response of a4 subtype-containing GABAA receptors to synaptic and extrasynaptic GABA. J. Physiol. 578, 655–676. Lambert, J.J., Peters, J.A., Sturgess, N.C., Hales, T.G., 1990. Steroid modulation of the GABAA receptor complex: electrophysiological studies. Ciba Found. Symp. 153, 56–71 (discussion 71–82). Lambert, J.J., Peters, J.A., Harvey, S., Belelli, D., 2001. Steroid modulation of GABAA receptors. In: Mohler, H. (Ed.), Pharmacology of Inhibitory Amino Acid Transmitters. Springer, pp. 117–140. Li, P., Covey, D.F., Steinbach, J.H., Akk, G., 2006. Dual potentiating and inhibitory actions of a benz[e]indene neurosteroid analog on recombinant a1b2g2 GABAA receptors. Mol. Pharmacol. 69, 2015–2026. Li, P., Bracamontes, J., Katona, B.W., Covey, D.F., Steinbach, J.H., Akk, G., 2007. Natural and enantiomeric etiocholanolone interact with distinct sites on the rat a1b2g2L GABAA receptor. Mol. Pharm. 71, 1582–1590. Luscher, B., Keller, C.A., 2004. Regulation of GABAA receptor trafficking, channel activity, and functional plasticity of inhibitory synapses. Pharmacol. Ther. 102, 195–221. Maguire, J., Mody, I., 2007. Neurosteroid synthesis-mediated regulation of GABAA receptors: relevance to the ovarian cycle and stress. J. Neurosci. 27, 2155–2162. Maguire, J.L., Stell, B.M., Rafizadeh, M., Mody, I., 2005. Ovarian cycle-linked changes in GABAA receptors mediating tonic inhibition alter seizure susceptibility and anxiety. Nat. Neurosci. 8, 797–804. Matsui, D., Sakari, M., Sato, T., Murayama, A., Takada, I., Kim, M., Takeyama, K., Kato, S., 2002. Transcriptional regulation of the mouse steroid 5areductase type II gene by progesterone in brain. Nucleic Acids Res. 30, 1387–1393. McHugh, T.J., Jones, M.W., Quinn, J.J., Balthasar, N., Coppari, R., Elmquist, J.K., Lowell, B.B., Fanselow, M.S., Wilson, M.A., Tonegawa, S., 2007. Dentate gyrus NMDA receptors mediate rapid pattern separation in the hippocampal network. Science 317, 94–99. Milligan, C.J., Buckley, N.J., Garret, M., Deuchars, J., Deuchars, S.A., 2004. Evidence for inhibition mediated by coassembly of GABAA and GABAC receptor subunits in native central neurons. J. Neurosci. 24, 7241– 7250. Mitev, Y.A., Darwish, M., Wolf, S.S., Holsboer, F., Almeida, O.F., Patchev, V.K., 2003. Gender differences in the regulation of 3a-hydroxysteroid dehydrogenase in rat brain and sensitivity to neurosteroid-mediated stress protection. Neuroscience 120, 541–549. Mohler, H., 2007. Molecular regulation of cognitive functions and developmental plasticity: impact of GABAA receptors. J. Neurochem. 102, 1–12. Morris, K.D., Amin, J., 2004. Insight into the mechanism of action of neuroactive steroids. Mol. Pharmacol. 66, 56–69.
E.A. Mitchell et al. / Neurochemistry International 52 (2008) 588–595 Nusser, Z., Mody, I., 2002. Selective modulation of tonic and phasic inhibitions in dentate gyrus granule cells. J. Neurophysiol. 87, 2624–2628. Nusser, Z., Sieghart, W., Stephenson, F.A., Somogyi, P., 1996. The a6 subunit of the GABAA receptor is concentrated in both inhibitory and excitatory synapses on cerebellar granule cells. J. Neurosci. 16, 103–114. Nusser, Z., Sieghart, W., Somogyi, P., 1998. Segregation of different GABAA receptors to synaptic and extrasynaptic membranes of cerebellar granule cells. J. Neurosci. 18, 1693–1703. Peters, J.A., Kirkness, E.F., Callachan, H., Lambert, J.J., Turner, A.J., 1988. Modulation of the GABAA receptor by depressant barbiturates and pregnane steroids. Br. J. Pharmacol. 94, 1257–1269. Pinna, G., Agis-Balboa, R.C., Doueiri, M.S., Guidotti, A., Costa, E., 2004. Brain neurosteroids in gender-related aggression induced by social isolation. Crit. Rev. Neurobiol. 16, 75–82. Poisbeau, P., Patte-Mensah, C., Keller, A.F., Barrot, M., Breton, J.D., LuisDelgado, O.E., Freund-Mercier, M.J., Mensah-Nyagan, A.G., Schlichter, R., 2005. Inflammatory pain upregulates spinal inhibition via endogenous neurosteroid production. J. Neurosci. 25, 11768–11776. Porcello, D.M., Huntsman, M.M., Mihalek, R.M., Homanics, G.E., Huguenard, J.R., 2003. Intact synaptic GABA-ergic inhibition and altered neurosteroid modulation of thalamic relay neurons in mice lacking d subunit. J. Neurophysiol. 89, 1378–1386. Pritchett, D.B., Seeburg, P.H., 1990. g-aminobutyric acidA receptor a5-subunit creates novel type II benzodiazepine receptor pharmacology. J. Neurochem. 54, 1802–1804. Pritchett, D.B., Sontheimer, H., Shivers, B.D., Ymer, S., Kettenmann, H., Schofield, P.R., Seeburg, P.H., 1989. Importance of a novel GABAA receptor subunit for benzodiazepine pharmacology. Nature 338, 582–585. Reddy, D.S., 2003. Is there a physiological role for the neurosteroid THDOC in stress-sensitive conditions? Trends Pharmacol. Sci. 24, 103–106. Represa, A., Ben Ari, Y., 2005. Trophic actions of GABA on neuronal development. Trends Neurosci. 28, 278–283. Reynolds, D.S., Rosahl, T.W., Cirone, J., O’Meara, G.F., Haythornthwaite, A., Newman, R.J., Myers, J., Sur, C., Howell, O., Rutter, A.R., Atack, J., Macaulay, A.J., Hadingham, K.L., Hutson, P.H., Belelli, D., Lambert, J.J., Dawson, G.R., McKernan, R., Whiting, P.J., Wafford, K.A., 2003. Sedation and anesthesia mediated by distinct GABAA receptor isoforms. J. Neurosci. 23, 8608–8617. Rick, C.E., Ye, Q., Finn, S.E., Harrison, N.L., 1998. Neurosteroids act on the GABAA receptor at sites on the N-terminal side of the middle of TM2. Neuroreport 9, 379–383. Rudolph, U., Mohler, H., 2006. GABA-based therapeutic approaches: GABAA receptor subtype functions. Curr. Opin. Pharmacol. 6, 18–23. Saxena, N.C., Macdonald, R.L., 1994. Assembly of GABAA receptor subunits: role of the d subunit. J. Neurosci. 14, 7077–7086. Schweizer, C., Balsiger, S., Bluethmann, H., Mansuy, I.M., Fritschy, J.M., Mohler, H., Luscher, B., 2003. The g2 subunit of GABAA receptors is required for maintenance of receptors at mature synapses. Mol. Cell. Neurosci. 24, 442–450.
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Semyanov, A., Walker, M.C., Kullmann, D.M., Silver, R.A., 2004. Tonically active GABAA receptors: modulating gain and maintaining the tone. Trends Neurosci. 27, 262–269. Shen, H., Gong, Q.H., Aoki, C., Yuan, M., Ruderman, Y., Dattilo, M., Williams, K., Smith, S.S., 2007. Reversal of neurosteroid effects at a4b2d GABAA receptors triggers anxiety at puberty. Nat. Neurosci. 10, 469–477. Sieghart, W., 1995. Structure and pharmacology of g-aminobutyric acidA receptor subtypes. Pharmacol. Rev. 47, 181–234. Sieghart, W., 2006. Structure, pharmacology, and function of GABAA receptor subtypes. Adv. Pharmacol. 54, 231–263. Sieghart, W., Sperk, G., 2002. Subunit composition, distribution and function of GABAA receptor subtypes. Curr. Top. Med. Chem. 2, 795–816. Sigel, E., Buhr, A., 1997. The benzodiazepine binding site of GABAA receptors. Trends Pharmacol. Sci. 18, 425–429. Stell, B.M., Brickley, S.G., Tang, C.Y., Farrant, M., Mody, I., 2003. Neuroactive steroids reduce neuronal excitability by selectively enhancing tonic inhibition mediated by delta subunit-containing GABAA receptors. Proc. Natl. Acad. Sci. U.S.A. 100, 14439–14444. Thomas, P., Mortensen, M., Hosie, A.M., Smart, T.G., 2005. Dynamic mobility of functional GABAA receptors at inhibitory synapses. Nat. Neurosci. 8, 889–897. Torres, J.M., Ortega, E., 2003. Differential regulation of steroid 5a-reductase isozymes expression by androgens in the adult rat brain. FASEB J. 17, 1428–1433. Ueno, S., Tsutsui, M., Toyohira, Y., Minami, K., Yanagihara, N., 2004. Sites of positive allosteric modulation by neurosteroids on ionotropic g-aminobutyric acid receptor subunits. FEBS Lett. 566, 213–217. Upasani, R.B., Yang, K.C., Acosta-Burruel, M., Konkoy, C.S., McLellan, J.A., Woodward, R.M., Lan, N.C., Carter, R.B., Hawkinson, J.E., 1997. 3aHydroxy-3b-(phenylethynyl)-5b-pregnan-20-ones: synthesis and pharmacological activity of neuroactive steroids with high affinity for GABAA receptors. J. Med. Chem. 40, 73–84. van Rijnsoever, C., Tauber, M., Choulli, M.K., Keist, R., Rudolph, U., Mohler, H., Fritschy, J.M., Crestani, F., 2004. Requirement of a5-GABAA receptors for the development of tolerance to the sedative action of diazepam in mice. J. Neurosci. 24, 6785–6790. Weir, C.J., Ling, A.T., Belelli, D., Wildsmith, J.A., Peters, J.A., Lambert, J.J., 2004. The interaction of anaesthetic steroids with recombinant glycine and GABAA receptors. Br. J. Anaesth. 92, 704–711. Wittmer, L.L., Hu, Y., Kalkbrenner, M., Evers, A.S., Zorumski, C.F., Covey, D.F., 1996. Enantioselectivity of steroid-induced g-aminobutyric acidA receptor modulation and anesthesia. Mol. Pharmacol. 50, 1581–1586. Wohlfarth, K.M., Bianchi, M.T., Macdonald, R.L., 2002. Enhanced neurosteroid potentiation of ternary GABAA receptors containing the d subunit. J. Neurosci. 22, 1541–1549. Wulff, P., Goetz, T., Leppa, E., Linden, A.M., Renzi, M., Swinny, J.D., Vekovischeva, O.Y., Sieghart, W., Somogyi, P., Korpi, E.R., Farrant, M., Wisden, W., 2007. From synapse to behavior: rapid modulation of defined neuronal types with engineered GABAA receptors. Nat. Neurosci. 10, 923–929.
Neurochemistry International 52 (2008) 588–595 www.elsevier.com/locate/neuint
Neurosteroid modulation of GABAA receptors: Molecular determinants and significance in health and disease Elizabeth A. Mitchell, Murray B. Herd, Benjamin G. Gunn, Jeremy J. Lambert, Delia Belelli * Neurosciences Institute, Division of Pathology and Neuroscience, University of Dundee, Ninewells Hospital and Medical School, Ninewells Hospital, Dundee DD1 9SY, United Kingdom Received 1 August 2007; received in revised form 30 September 2007; accepted 10 October 2007 Available online 16 October 2007
Abstract Over the past 20 years it has become apparent that certain steroids, synthesised de novo in the brain, hence named neurosteroids, produce immediate changes (within seconds) in neuronal excitability, a time scale that precludes a genomic locus of action. Identified molecular targets underlying modulation of brain excitability include both the inhibitory GABAA and the excitatory NMDA receptor. Of particular interest is the interaction of certain neurosteroids with the GABAA receptor, the major inhibitory receptor in mammalian brain. During the last decade, compelling evidence has accrued to reveal that locally produced neurosteroids may selectively ‘‘fine tune’’ neuronal inhibition. A range of molecular mechanisms including the subunit composition of the receptor(s), phosphorylation and local steroid metabolism, underpin the regionand neuronal selectivity of action of neurosteroids at synaptic and extrasynaptic GABAA receptors. The relative contribution played by each of these mechanisms in a variety of physiological and pathophysiological scenarios is currently being scrutinised at a cellular and molecular level. However, it is not known how such mechanisms may act in concert to influence behavioural profiles in health and disease. An important question concerns the identification of the anatomical substrates mediating the repertoire of behaviours produced by neurosteroids. ‘‘Knock-in’’ mice expressing mutant GABAA subunits engineered to be insensitive to benzodiazepines or general anaesthetics have proved invaluable in evaluating the role of GABAA receptor subtypes in complex behaviours such as sedation, cognition and anxiety [Rudolph, U., Mohler, H., 2006. GABA-based therapeutic approaches: GABAA receptor subtype functions. Curr. Opin. Pharmacol. 6, 18–23]. However, the development of a similar approach for neurosteroids has been hampered by the limited knowledge that, until recently, has surrounded the identity of the amino acid residues contributing to the neurosteroid binding pocket. Here, we will review recent progress in identifying the neurosteroid binding site on the GABAA receptor, and discuss how these discoveries will impact on our understanding of the role of neurosteroids in health and disease. # 2007 Elsevier Ltd. All rights reserved. Keywords: Neurosteroid binding site; GABAA receptor delta subunit; Extrasynaptic conductance; Anxiolytic; ‘‘Knock-in’’ mice
1. Introduction The neurotransmitter GABA, acting through GABAA receptors, mediates the majority of ‘‘fast’’ inhibitory synaptic signalling in the mammalian central nervous system (CNS) and consequently has a profound influence on our mood and behaviour. Additionally, it has recently been recognized that GABAA receptors underpin a sustained, tonic form of inhibition via extrasynaptically located receptors, which play a significant
* Corresponding author. Tel.: +44 1382 632161; fax: +44 1382 667120. E-mail address: [email protected] (D. Belelli). 0197-0186/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2007.10.007
role in brain excitability, both in normal and pathophysiological states (Semyanov et al., 2004; Farrant and Nusser, 2005). GABAA receptors are an important target for a variety of clinically important drugs including the benzodiazepines (used in the treatment of anxiety, epilepsy, sleep disorders and alcohol withdrawal, etc.) and a number of general anaesthetics such as propofol, etomidate and thiopental that are used to induce anaesthesia (Sieghart, 1995; Sigel and Buhr, 1997; Belelli et al., 1999; Rudolph and Mohler, 2006). These compounds all act as positive allosteric modulators to potentiate inhibition mediated by GABAA receptors. A variety of neurological and psychiatric disorders, which include epilepsy, pain syndromes, anxiety, depression and schizophrenia exhibit perturbations of GABAergic transmission. Interestingly, some of these conditions have
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been associated with abnormal levels of certain endogenously occurring neurosteroids, which potently and selectively enhance the function of the GABAA receptor via a distinct site (reviewed in Belelli and Lambert, 2005; Akk et al., 2007; Herd et al., 2007; Hosie et al., 2007). Neurosteroids, typified by the potent progesterone metabolite, 5a-pregnan-3a-ol-20-one (3a,5a-THPROG) can be synthesised de novo in the CNS, both in neurons and glia in levels sufficient to modulate GABAA receptor function and thus, are likely to play an important physiological/pathophysiological role (Belelli and Lambert, 2005; Herd et al., 2007). Indeed, a direct link between abnormal neurosteroid synthesis and brain function has been documented together with the demonstration of sex differences in the genomic regulation of the enzymes responsible for their synthesis and degradation (Matsui et al., 2002; Mitev et al., 2003; Reddy, 2003; Torres and Ortega, 2003; Pinna et al., 2004). Furthermore, concomitant with the demonstration of the trophic actions of GABA in the developing CNS (e.g. in neuronal proliferation, differentiation, migration and synapse establishment; Represa and Ben Ari, 2005), a role for neurosteroids in neurodevelopment has begun to emerge (Grobin et al., 2006). Although a persuasive body of indirect evidence has accumulated over the past 20 years to support the existence of a specific neurosteroid binding site(s) on the receptor protein, attempts to identify such a site(s) have proved, until lately, unsuccessful. Recently, the application of molecular modelling of chimaeric constructs made of steroid-sensitive and insensitive GABA subunits has identified putative neurosteroid binding sites and revealed specific amino acid residues that govern the steroid/receptor interaction. The molecular mechanisms underpinning the selectivity of steroid action at synaptic and extrasynaptic GABAA receptors have been reviewed recently (Herd et al., 2007). Here, we specifically consider advances in defining putative neurosteroid binding pockets on the GABAA receptor protein. We then speculate on how this new information may be exploited to advance our understanding of the role of neurosteroids in health and disease. 2. GABAA receptor heterogeneity Advances in our understanding of neurosteroids are in part intrinsically linked to progress in elucidating the complex roles different GABAA receptor isoforms play in neuronal signalling, development and behaviour. The GABAA receptor is a member of the Cys-loop family of transmitter-gated ion channels and is composed of five trans-membrane subunits arranged to form an intrinsic anion-conducting channel. The cloning of the first subunits (a1 and b1) in the late 1980s led to the subsequent discovery of a further 17 subunits (a1–6, b1–3, g1–3, d, e, u, p and r1–3) classed according to sequence homology (70% within a subunit class, 30% between classes), a heterogeneity further increased by alternative exon splicing (Barnard et al., 1998; Sieghart, 2006). In the absence of rules governing subunit co-assembly, this repertoire could theoretically underpin the existence of thousands of distinct
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GABAA receptor subtypes. However, strict subunit assembly, trafficking requirements and region-selective subunit expression patterns (Sieghart and Sperk, 2002; Fritschy and Brunig, 2003; Luscher and Keller, 2004), refine this number to 30 main GABAA receptor subtypes, each with distinct physiological and pharmacological properties. The majority of GABAA receptors in vivo are composed of a, b, and g subunits, in a probable stoichiometry of 2a, 2b to 1g subunit. By far the most abundant subtype is the a1b2g2 isoform (60%) with a2b3g2 and a3bxg2 representing 15–20% and 10–15% of the total, respectively (Mohler, 2007). Receptors incorporating a4, a5, a6, b1, g1, g3, d, e, and u subunits are less numerous, but nevertheless serve important functions. The d and e subunits appear to substitute for the g subunit and receptor isoforms incorporating the d subunit specifically localise to extrasynaptic/perisynaptic regions in selective neuronal populations (e.g. cerebellar and dentate gyrus granule cells, thalamic relay nuclei and neocortex; Nusser et al., 1996, 1998; Brickley et al., 2001; Nusser and Mody, 2002; Stell et al., 2003; Belelli et al., 2005; Cope et al., 2005; Chandra et al., 2006; Drasbek and Jensen, 2006; Bright et al., 2007). The r subunits form both homomeric and heteromeric GABA-gated chloride channels with distinct pharmacological properties leading some researchers to separately characterise these receptors as GABAC. However, evidence that, in vivo, these subunits may co-assemble with members of the GABAA receptor subunit class may warrant, in the near future, their inclusion in the GABAA receptor category (Milligan et al., 2004; Harvey et al., 2006). The subunit composition influences fundamental features of the receptor including the sensitivity to GABA, channel kinetics, desensitisation, pharmacological properties and the neuronal location (e.g. synaptic/extrasynaptic). Thus, as noted above, receptors incorporating the d subunit, in combination with a b and, usually, either an a4, a6 or a1 subunit, are insensitive to modulation by benzodiazepines and localise at extrasynaptic sites, where they mediate a persistent form of tonic inhibition. The properties of d-containing GABAA receptors are tailored for such a role as they exhibit a relatively high affinity for GABA and limited desensitisation, features that permit a maintained response to ambient GABA (Saxena and Macdonald, 1994; Haas and Macdonald, 1999; Bianchi et al., 2002; Lagrange et al., 2007). By contrast, synaptically located receptors typically incorporate the g2 subunit in combination with different a and b subunit isoforms. In agreement, the g2 subunit is crucial for receptor clustering and synaptic localisation (Essrich et al., 1998; Fritschy and Brunig, 2003; Schweizer et al., 2003). However, g2-containing receptors may additionally localise at extrasynaptic locations (e.g. a3b2/3g2 or a5b3g2; see Devor et al., 2001; Caraiscos et al., 2004). Indeed, g2-containing synaptic GABAA receptors appear to be directly recruited, by lateral diffusion, from a dynamic pool of extrasynaptic GABAA receptors (Thomas et al., 2005; Bogdanov et al., 2006). The pharmacological and behavioural relevance of GABAA receptor subtypes has been elegantly illustrated by the
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behavioural analysis of transgenic mouse models (i.e. ‘‘knockin’’ mice). Such models were developed subsequent to the identification of specific residues within a and b subunits that govern the actions of benzodiazepines and the general anaesthetic etomidate, respectively. This approach has enabled a genetic dissection of the behavioural repertoire associated with the systemic administration of such agents. Thus, for example, the sedative, amnestic and a component of the anticonvulsant effects of diazepam are mediated by receptors incorporating the a1 subunit, whereas the anxiolytic and myorelaxant actions of this benzodiazepine implicate a role for a2-GABAA receptors with a contribution from a3 and a5 subunit containing receptors, respectively (van Rijnsoever et al., 2004; Mohler, 2007). A similar approach has characterised the contribution of GABAA receptor subtypes incorporating different b subunit isoforms to the variety of behaviours associated with general anaesthetics, e.g. the sedative and immobilising actions of etomidate are mediated by b2- and b3-containing GABAA receptors, respectively (Jurd et al., 2003; Reynolds et al., 2003). Whether similar receptor isoforms segregate with the behavioural repertoire associated with neurosteroids remains to be determined. In this context, of specific interest is the role of the benzodiazepine-insensitive dcontaining extrasynaptic GABAA receptors, which have been proposed to represent a significant molecular target underpinning many of the physiologically relevant actions of neurosteroids (e.g. anxiolytic and anticonvulsant effects; Maguire et al., 2005, see below). 3. A distinct neurosteroid binding site 3.1. Indirect evidence Although the GABAA receptor-active neurosteroids such as 3a,5a-THPROG, are highly lipid soluble (oil to water coefficient > 10,000), their potent and stereoselective interaction with native GABAA receptors suggested a specific neurosteroid recognition site on the receptor protein (Callachan et al., 1987; Harrison et al., 1987; Peters et al., 1988). This concept was bolstered by the subsequent observation that GABAA receptor-active steroids exhibit a clear enantioselectivity, both in vitro and in vivo (Wittmer et al., 1996), a finding indicative of a selective interaction within a chiral environment, traditionally afforded by protein structures. In agreement, the effects of two pairs of anaesthetic steroid enantiomers upon lipid bilayers lack enantiomeric specificity (Alakoskela et al., 2007). Early radioligand binding and electrophysiological studies provided unambiguous evidence that the recognition site for neurosteroids was distinct from other known allosteric binding sites (e.g. benzodiazepine and barbiturates) on the GABAA receptor (Callachan et al., 1987; Gee et al., 1988; Peters et al., 1988). The discovery that neurosteroids could enhance GABAevoked responses at low nanomolar concentrations and directly activate the receptor in the submicromolar range, postulated the existence of at least two distinct binding sites (Callachan et al., 1987). However, until recently, the identification of such sites has remained elusive.
Traditional approaches aimed at identifying binding sites on receptor proteins have typically relied on the use of photolabelling agents and/or functional analysis of chimaeric constructs between ligand-sensitive and -insensitive subunits. Such methods have revealed residues that contribute to the binding pocket for benzodiazepines (Pritchett et al., 1989; Pritchett and Seeburg, 1990; Duncalfe et al., 1996; reviewed in Sigel and Buhr, 1997). Despite the potency inferred by functional studies, neurosteroid lipophilicity has hampered the development of suitable radioligands. The pregnane neurosteroids are highly selective for the GABAA receptor. However, in contrast to the exquisite subunit selectivity exhibited by benzodiazepines, recombinant expression studies have revealed a promiscuous interaction of neurosteroids with many GABAA receptor isoforms (Belelli et al., 2002). This lack of specificity has confounded the use of chimaeric constructs and mutagenesis (Rick et al., 1998; Akk et al., 2004; Ueno et al., 2004, see below). Recently, an investigation into how neurosteroids access the receptor has been pursued as an indirect approach to gain an insight into the location of the neurosteroid binding site(s) (Akk et al., 2005). Utilising the cell-attached configuration of the patch clamp technique, Akk and colleagues demonstrated that aqueous extracellular access is not a prerequisite for neurosteroid modulation of the GABAA receptor. Specifically, they revealed that the enhancement of GABA-gated channel activity produced when 3a,5a-THPROG is included in the patch pipette is indistinguishable from that caused by the bathapplication of the steroid. Furthermore, tracking the steroid access route with a fluorescent derivative of 3a,5a-THPROG, revealed prominent steroid accumulation, both in the plasma membrane and intracellularly. These findings are consistent with a route of access through the membrane, a conclusion strengthened by the observation that a membrane-impermeant steroid analogue enhances channel activity only when applied to the inner, but not to the outer, membrane leaflet (Akk et al., 2005). Collectively, these recent observations support earlier proposals (Lambert et al., 1990) that the interaction of neurosteroids with GABAA receptors may be mediated via a membrane-embedded hydrophobic pocket of the receptor protein, presumably accessed by the steroid via lateral diffusion. 3.2. Identification of the neurosteroid binding pocket As noted above, the limited selectivity exhibited by neurosteroids for different GABAA receptor isoforms has complicated identification of their binding site(s). However, the closely related glycine receptor is insensitive to such steroids, including the synthetic anaesthetic steroid alphaxalone (Harrison et al., 1987; Weir et al., 2004). Functional analysis of chimaeras composed of alphaxalone-sensitive GABAA receptor a2 or b1 subunits and the alphaxalone-insensitive glycine receptor a1 subunit, suggested a binding domain located in a region N-terminal to the TM2 domain of the a2 and/ or b1 subunit (Rick et al., 1998). Subsequent attempts employed chimaeric constructs between the glycine a1 (Ueno
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et al., 2004), or GABA r subunit (Akk et al., 2004) and the GABAA receptor a1 or g2 subunit, respectively. However, functional analysis of these chimaeras implicated a different (TM4) trans-membrane portion of the receptor protein, from that suggested by Rick et al. (1998). Nonetheless, collectively, such reports highlighted the importance of the trans-membrane regions of the protein for neurosteroid action, but did not unambiguously pinpoint their location, nor did these studies identify the residues forming the binding pocket. We and others had previously demonstrated that a GABAgated chloride channel derived from Drosophila (RDL) was relatively insensitive to neurosteroids (Chen et al., 1994; Belelli et al., 1996; Hosie and Sattelle, 1996). Recently, chimaeric constructs of RDL and mammalian GABAA subunits were used to generate neurosteroid-insensitive GABAA receptors (Hosie et al., 2006). Incorporation of the TM1 to TM2 region of the RDL GABA subunit into the murine a1, but not the b2 subunit, proved sufficient to confer insensitivity to the GABAenhancing and ‘‘agonist’’ actions of 3a,5a-THPROG and 3a,5a-tetrahydrodeoxycorticosterone (3a,5a-THDOC) determined with a heterologously expressed ternary aRDL-b-g receptor complex (Hosie et al., 2006). This advance, coupled with a retrospective re-evaluation of the steroid structure activity requirements for modulation of GABAA receptor function has proved invaluable to identifying specific amino acid residues that may contribute to the neurosteroid binding pocket. The critical importance for GABAA receptor activity of a hydroxyl and a ketone group in the C3a and C20 positions, respectively (Gee et al., 1987, 1988; Harrison et al., 1987;
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Peters et al., 1988; Lambert et al., 2001), suggested that neurosteroids would specifically interact with suitably spaced hydrophilic residues within the receptor protein. In addition, early models of the neurosteroid pharmacophore had postulated the hydrophobic nature of the four rings of the steroid backbone to be sufficient to allow access to a hydrophobic pocket within the protein (Upasani et al., 1997). The hydrogen bonding acceptor and donor properties afforded by the 3a-hydroxyl and 20-ketone groups, respectively, would provide the primary ‘‘docking points’’ necessary to confer a high degree of potency, specificity and efficacy to the neurosteroid interaction with the GABAA receptor (Upasani et al., 1997). The search for the polar, or charged residues that would allow the formation of such hydrogen bonds within the TM1–TM2 domains of the mouse a1 subunit resulted in the specific identification of threonine 236 and glutamine 241. These residues are crucial determinants of the ‘‘agonist’’ and the GABA-enhancing actions, respectively of both 3a,5a-THPROG and 3a,5aTHDOC. 3.3. Steroid-sensitive and -insensitive GABA subunits Consistent with the putative role played by residues threonine 236 and glutamine 241 in the formation of hydrogen bonds with GABAA receptor-active steroid molecules, hydrophobic/non-polar residues occupy equivalent positions in steroid-insensitive subunits (Fig. 1). Thus, a non-polar isoleucine, or methionine residue occupies the homologous position to threonine 236 in the equivalent position of the glycine a1/b and GABA r subunit, respectively. Receptors
Fig. 1. Sequence alignment of trans-membrane region (TM1) of the Drosophila RDL, human (h) GABAA, rat (r) and human glycine receptor subunits. Amino acid residues important for the GABA-enhancing (residue 241) and agonist (residue 236) actions of 3a,5a-THPROG and 3a,5a-THDOC at GABAA receptors are coloured grey for RDL, GABAA and glycine receptor subunits.
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Fig. 2. Sequence alignment of the COOH-terminal region including trans-membrane region (TM4), of the Drosophila RDL, human (h) GABAA, human and rat (r) glycine receptor subunits. Amino acid residues (407 and 410), which additionally contribute to the binding pocket mediating the GABA-enhancing actions of 3a,5aTHPROG and 3a,5a-THDOC at GABAA receptors, are coloured grey for RDL, GABAA and glycine receptor subunits.
formed from these subunits are insensitive to the actions of 5areduced steroids (Belelli et al., 1999; Weir et al., 2004; Morris and Amin, 2004). Similarly, while glutamine 241 is conserved across all the a subunit isoforms (Fig. 1), replacement of this residue by tryptophan (the residue that occupies the equivalent position of the RDL subunit) abolishes enhancement of the GABA-evoked response by the steroid (Hosie et al., 2006), probably because this aromatic residue can only act to provide hydrogen bonds. Interestingly, a tryptophan residue also occupies the homologous position within the steroid-insensitive glycine a1/b and GABA r subunits (Fig. 2). Clearly, it would be of interest to determine whether replacement of this tryptophan residue with glutamine is sufficient to impart steroid sensitivity to either the glycine a1/b, or GABA r subunits. As the C20 ketone group can only act as a hydrogen bond acceptor, collectively, these findings indicate the glutamine residue to be a crucial determinant, allowing a hydrogen bond-mediated interaction of the a subunit with the 3a hydroxyl group. Subsequent molecular modelling of the binding site, utilising the calculated distance between the two putative docking centres of the steroid molecule as a guide (i.e. the C3 hydroxyl and the C20 ketone groups), has uncovered an additional contribution made by two residues located in the TM4 domain (N407 and Y410). These amino acids appear to contribute to the proposed pocket that mediates the GABA-modulatory actions of the neurosteroids. Interestingly, the residues are conserved across the steroidsensitive and -insensitive subunits (see Fig. 2). Collectively, these findings are consistent with a contribution by the three residues to a single potentiating neurosteroid site. However, the recent demonstration that the potentiating actions of two steroids (5b-3a-hydroxy-18-norandrostane17b-carbonitrile, and the unnatural enantiomer of etiocholanolone) are not similarly influenced by mutations of these residues complicates the interpretation of the above findings
and raises the prospect of multiple steroid recognition sites (Li et al., 2006, 2007). 3.4. Implications for the putative physiological/ pathophysiological role of neurosteroids The molecular identification of the residues contributing to the sites mediating the modulatory and agonist actions of neurosteroids has finally provided the information required to allow the creation of a generation of subunit specific neurosteroid-insensitive ‘‘knock-in’’ mice. Such mice should permit a novel insight into the proposed physiological and pathophysiological roles of neurosteroids and identify which GABAA receptors mediate these steroid-induced behaviours. However, the quest to identify the neuronal substrates of neurosteroid actions in health and disease poses several challenges. Importantly, the choice of a suitable amino acid substitution, which, while conferring insensitivity to the action of neurosteroids such as 3a,5a-THPROG and 3a,5a-THDOC, should produce little or no change to the receptor sensitivity to the neurotransmitter GABA. Indeed, mice harbouring mutations that not only modify drug binding properties, but additionally alter the receptor sensitivity to GABA, may exhibit behavioural abnormalities that complicate interpretation (Homanics et al., 2005). Importantly, the glutamine to leucine 241 mutation of the a subunit abolished neurosteroid enhancement of the GABA response, but in contrast to the glutamine to tryptophan mutation, the modification additionally had a modest effect on the interaction of GABA with the receptor (Hosie et al., 2006). Although such mutations appear to have only a modest influence on recombinant GABAA receptor function, their impact on the expression, trafficking and function of native neuronal GABAA receptors will need to be verified in the appropriate ‘‘knock-in’’ mice.
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Clearly, inherent in the hypothesis that neurosteroids may physiologically influence GABAA receptor-mediated inhibition is the prediction of a behavioural phenotype, which presumably will be dependent on the a subunit isoform that is ‘‘steroidinactivated’’. To unequivocally elucidate the role played by neurosteroids in specific brain functions may require a reversible and temporally controlled method to ‘‘silence’’ the steroid sensitivity of a specific receptor isoform in a defined neuronal population (Wulff et al., 2007). Such an approach would offer the obvious advantage of overcoming the complications (e.g. compensations) arising from irreversible genetic alterations (Brickley et al., 2001) and additionally may permit the putative role of neurosteroids in neurodevelopment to be explored more meaningfully. Although the mammalian CNS expresses a palette of 20–30 major GABAA receptor subtypes, physiologically a more restricted number of receptors may be influenced by endogenous neurosteroid levels. As the ‘‘silencing’’ mutation would be introduced into the a subunit, the question arises as to which receptor populations to target. Extrasynaptic d-containing GABAA receptors, particularly those incorporating the a4 subunit, may mediate many of the behavioural actions of neurosteroids that occur for example during the ovarian cycle, stress and puberty (Maguire et al., 2005; Maguire and Mody, 2007; Shen et al., 2007). Recombinant expression studies unanimously support a preferential interaction of neurosteroids with d-containing receptor isoforms (Belelli et al., 2002; Brown et al., 2002; Wohlfarth et al., 2002), although investigations of the in vitro actions of neurosteroids such as 3a,5a-THPROG and 3a,5aTHDOC in a native environment have produced apparently inconsistent findings. Indeed, not all native d-containing extrasynaptic GABAA receptors are sensitive to low, physiological concentrations of neurosteroids (e.g. thalamic tonic conductance, Porcello et al., 2003, reviewed by Herd et al., 2007). Therefore, should a loss of steroid sensitivity be demonstrated in recombinant a4Q241Wbd GABAA receptors, investigation of steroid action in neurones putatively expressing this receptor isoform could provide an interesting ‘‘proof of concept’’ study. Ultimately, this approach could be further refined to restrict the expression of the mutant subunit to a defined neuronal population (e.g. dentate gyrus granule cells, McHugh et al., 2007; Wulff et al., 2007). Such models would permit the contribution of specific neuronal receptor populations to complex neurosteroid-induced behaviours to be dissected. However, extrasynaptic GABAA receptors may not be the only significant molecular target of neurosteroid actions. Indeed, current evidence suggests that modulation of synaptic GABAA receptors may play an equally important role. Thus, for example, synaptic a3b3g2 GABAA receptors in the spinal cord may mediate the putative endogenous analgesic actions of neurosteroids (Poisbeau et al., 2005). Clearly, the creation of mice rendered steroid-insensitive at the a3-containing GABAA receptors in neuronal populations associated with the processing of nociceptive signals (e.g. spinal cord) may be particularly instructive in defining the putative role played by neurosteroids as analgesic agents.
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4. Conclusions Neurosteroids, typified by 3a,5a-THPROG and 3a,5aTHDOC are now recognized as highly selective and potent endogenous modulators of GABAA receptor-mediated synaptic and extrasynaptic neurotransmission. The recent identification of specific residues that contribute to the neurosteroid recognition site(s) is an essential precursor to the generation of mice that express steroid-insensitive GABAA receptor isoforms. Such models should permit the physiological and pathophysiological effects of neurosteroids to be dissected in a manner analogous to benzodiazepines and general anaesthetics. The selective interaction of neurosteroids with neuronal GABAA receptors is influenced by the subunit composition of the receptor, local metabolism and phosphorylation. Therefore, this approach may provide a unique opportunity to investigate how these different mechanisms act in concert to influence neurosteroid regulation of brain excitability in health and disease. Acknowledgements Some aspects of the work described here were supported by the MRC UK, a BBSRC Project Grant and Tenovus Scotland. We thank Dr. Michelle Cooper for providing the sequence alignments. References Akk, G., Bracamontes, J.R., Covey, D.F., Evers, A., Dao, T., Steinbach, J.H., 2004. Neuroactive steroids have multiple actions to potentiate GABAA receptors. J. Physiol. 558, 59–74. Akk, G., Shu, H.J., Wang, C., Steinbach, J.H., Zorumski, C.F., Covey, D.F., Mennerick, S., 2005. Neurosteroid access to the GABAA receptor. J. Neurosci. 25, 11605–11613. Akk, G., Covey, D.F., Evers, A.S., Steinbach, J.H., Zorumski, C.F., Mennerick, S., 2007. Mechanisms of neurosteroid interactions with GABAA receptors. Pharmacol. Ther. 116, 35–37. Alakoskela, J.M., Covey, D.F., Kinnunen, P.K., 2007. Lack of enantiomeric specificity in the effects of anesthetic steroids on lipid bilayers. Biochim. Biophys. Acta 1768, 131–145. Barnard, E.A., Skolnick, P., Olsen, R.W., Mohler, H., Sieghart, W., Biggio, G., Braestrup, C., Bateson, A.N., Langer, S.Z., 1998. International Union of Pharmacology. XV. Subtypes of gamma-aminobutyric acidA receptors: classification on the basis of subunit structure and receptor function. Pharmacol. Rev. 50, 291–313. Belelli, D., Lambert, J.J., 2005. Neurosteroids: endogenous regulators of the GABAA receptor. Nat. Rev. Neurosci. 6, 565–575. Belelli, D., Callachan, H., Hill-Venning, C., Peters, J.A., Lambert, J.J., 1996. Interaction of positive allosteric modulators with human and Drosophila recombinant GABA receptors expressed in Xenopus laevis oocytes. Br. J. Pharmacol. 118, 563–576. Belelli, D., Pistis, M., Peters, J.A., Lambert, J.J., 1999. General anaesthetic action at transmitter-gated inhibitory amino acid receptors. Trends Pharmacol. Sci. 20, 496–502. Belelli, D., Casula, A., Ling, A., Lambert, J.J., 2002. The influence of subunit composition on the interaction of neurosteroids with GABAA receptors. Neuropharmacology 43, 651–661. Belelli, D., Peden, D.R., Rosahl, T.W., Wafford, K.A., Lambert, J.J., 2005. Extrasynaptic GABAA receptors of thalamocortical neurons: a molecular target for hypnotics. J. Neurosci. 25, 11513–11520. Bianchi, M.T., Haas, K.F., Macdonald, R.L., 2002. a1 and a6 subunits specify distinct desensitization, deactivation and neurosteroid modulation of
594
E.A. Mitchell et al. / Neurochemistry International 52 (2008) 588–595
GABAA receptors containing the d subunit. Neuropharmacology 43, 492–502. Bogdanov, Y., Michels, G., Armstrong-Gold, C., Haydon, P.G., Lindstrom, J., Pangalos, M., Moss, S.J., 2006. Synaptic GABAA receptors are directly recruited from their extrasynaptic counterparts. EMBO J. 25, 4381–4389. Brickley, S.G., Revilla, V., Cull-Candy, S.G., Wisden, W., Farrant, M., 2001. Adaptive regulation of neuronal excitability by a voltage-independent potassium conductance. Nature 409, 88–92. Bright, D.P., Aller, M.I., Brickley, S.G., 2007. Synaptic release generates a tonic GABAA receptor-mediated conductance that modulates burst precision in thalamic relay neurons. J. Neurosci. 27, 2560–2569. Brown, N., Kerby, J., Bonnert, T.P., Whiting, P.J., Wafford, K.A., 2002. Pharmacological characterization of a novel cell line expressing human a4b3d GABAA receptors. Br. J. Pharmacol. 136, 965–974. Callachan, H., Cottrell, G.A., Hather, N.Y., Lambert, J.J., Nooney, J.M., Peters, J.A., 1987. Modulation of the GABAA receptor by progesterone metabolites. Proc. R. Soc. Lond. B: Biol. Sci. 231, 359–369. Caraiscos, V.B., Elliott, E.M., You, T., Cheng, V.Y., Belelli, D., Newell, J.G., Jackson, M.F., Lambert, J.J., Rosahl, T.W., Wafford, K.A., MacDonald, J.F., Orser, B.A., 2004. Tonic inhibition in mouse hippocampal CA1 pyramidal neurons is mediated by a5 subunit-containing g-aminobutyric acid type A receptors. Proc. Natl. Acad. Sci. U.S.A. 101, 3662–3667. Chandra, D., Jia, F., Liang, J., Peng, Z., Suryanarayanan, A., Werner, D.F., Spigelman, I., Houser, C.R., Olsen, R.W., Harrison, N.L., Homanics, G.E., 2006. GABAA receptor a4 subunits mediate extrasynaptic inhibition in thalamus and dentate gyrus and the action of gaboxadol. Proc. Natl. Acad. Sci. U.S.A. 103, 15230–15235. Chen, R., Belelli, D., Lambert, J.J., Peters, J.A., Reyes, A., Lan, N.C., 1994. Cloning and functional expression of a Drosophila g-aminobutyric acid receptor. Proc. Natl. Acad. Sci. U.S.A. 91, 6069–6073. Cope, D.W., Hughes, S.W., Crunelli, V., 2005. GABAA receptor-mediated tonic inhibition in thalamic neurons. J. Neurosci. 25, 11553–11563. Devor, A., Fritschy, J.M., Yarom, Y., 2001. Spatial distribution and subunit composition of GABAA receptors in the inferior olivary nucleus. J. Neurophysiol. 85, 1686–1696. Drasbek, K.R., Jensen, K., 2006. THIP, a hypnotic and antinociceptive drug, enhances an extrasynaptic GABAA receptor-mediated conductance in mouse neocortex. Cereb. Cortex 16, 1134–1141. Duncalfe, L.L., Carpenter, M.R., Smillie, L.B., Martin, I.L., Dunn, S.M., 1996. The major site of photoaffinity labeling of the g-aminobutyric acid type A receptor by [3H]flunitrazepam is histidine 102 of the a subunit. J. Biol. Chem. 271, 9209–9214. Essrich, C., Lorez, M., Benson, J.A., Fritschy, J.M., Luscher, B., 1998. Postsynaptic clustering of major GABAA receptor subtypes requires the gamma 2 subunit and gephyrin. Nat. Neurosci. 1, 563–571. Farrant, M., Nusser, Z., 2005. Variations on an inhibitory theme: phasic and tonic activation of GABAA receptors. Nat. Rev. Neurosci. 6, 215–229. Fritschy, J.M., Brunig, I., 2003. Formation and plasticity of GABA-ergic synapses: physiological mechanisms and pathophysiological implications. Pharmacol. Ther. 98, 299–323. Gee, K.W., Chang, W.C., Brinton, R.E., McEwen, B.S., 1987. GABA-dependent modulation of the Cl-ionophore by steroids in rat brain. Eur. J. Pharmacol. 136, 419–423. Gee, K.W., Bolger, M.B., Brinton, R.E., Coirini, H., McEwen, B.S., 1988. Steroid modulation of the chloride ionophore in rat brain: structure-activity requirements, regional dependence and mechanism of action. J. Pharmacol. Exp. Ther. 246, 803–812. Grobin, A.C., Gizerian, S., Lieberman, J.A., Morrow, A.L., 2006. Perinatal allopregnanolone influences prefrontal cortex structure, connectivity and behavior in adult rats. Neuroscience 138, 809–819. Haas, K.F., Macdonald, R.L., 1999. GABAA receptor subunit g2 and d subtypes confer unique kinetic properties on recombinant GABAA receptor currents in mouse fibroblasts. J. Physiol. 514 (Pt 1), 27–45. Harrison, N.L., Majewska, M.D., Harrington, J.W., Barker, J.L., 1987. Structure-activity relationships for steroid interaction with the g-aminobutyric acidA receptor complex. J. Pharmacol. Exp. Ther. 241, 346–353.
Harvey, V.L., Duguid, I.C., Krasel, C., Stephens, G.J., 2006. Evidence that GABA r subunits contribute to functional ionotropic GABA receptors in mouse cerebellar Purkinje cells. J. Physiol. 577, 127–139. Herd, M.B., Belelli, D., Lambert, J.J., 2007. Neurosteroid modulation of synaptic and extrasynaptic GABAA receptors. Pharmacol. Ther. 116, 20–34. Homanics, G.E., Elsen, F.P., Ying, S.W., Jenkins, A., Ferguson, C., Sloat, B., Yuditskaya, S., Goldstein, P.A., Kralic, J.E., Morrow, A.L., Harrison, N.L., 2005. A gain-of-function mutation in the GABA receptor produces synaptic and behavioral abnormalities in the mouse. Genes Brain Behav. 4, 10–19. Hosie, A.M., Sattelle, D.B., 1996. Allosteric modulation of an expressed homooligomeric GABA-gated chloride channel of Drosophila melanogaster. Br. J. Pharmacol. 117, 1229–1237. Hosie, A.M., Wilkins, M.E., da Silva, H.M., Smart, T.G., 2006. Endogenous neurosteroids regulate GABAA receptors through two discrete transmembrane sites. Nature 444, 486–489. Hosie, A.M., Wilkins, M.E., Smart, T.G., 2007. Neurosteroid binding sites on GABAA receptors. Pharmacol. Ther. 116, 7–19. Jurd, R., Arras, M., Lambert, S., Drexler, B., Siegwart, R., Crestani, F., Zaugg, M., Vogt, K.E., Ledermann, B., Antkowiak, B., Rudolph, U., 2003. General anesthetic actions in vivo strongly attenuated by a point mutation in the GABAA receptor b3 subunit. FASEB J. 17, 250–252. Lagrange, A.H., Botzolakis, E.J., Macdonald, R.L., 2007. Enhanced macroscopic desensitization shapes the response of a4 subtype-containing GABAA receptors to synaptic and extrasynaptic GABA. J. Physiol. 578, 655–676. Lambert, J.J., Peters, J.A., Sturgess, N.C., Hales, T.G., 1990. Steroid modulation of the GABAA receptor complex: electrophysiological studies. Ciba Found. Symp. 153, 56–71 (discussion 71–82). Lambert, J.J., Peters, J.A., Harvey, S., Belelli, D., 2001. Steroid modulation of GABAA receptors. In: Mohler, H. (Ed.), Pharmacology of Inhibitory Amino Acid Transmitters. Springer, pp. 117–140. Li, P., Covey, D.F., Steinbach, J.H., Akk, G., 2006. Dual potentiating and inhibitory actions of a benz[e]indene neurosteroid analog on recombinant a1b2g2 GABAA receptors. Mol. Pharmacol. 69, 2015–2026. Li, P., Bracamontes, J., Katona, B.W., Covey, D.F., Steinbach, J.H., Akk, G., 2007. Natural and enantiomeric etiocholanolone interact with distinct sites on the rat a1b2g2L GABAA receptor. Mol. Pharm. 71, 1582–1590. Luscher, B., Keller, C.A., 2004. Regulation of GABAA receptor trafficking, channel activity, and functional plasticity of inhibitory synapses. Pharmacol. Ther. 102, 195–221. Maguire, J., Mody, I., 2007. Neurosteroid synthesis-mediated regulation of GABAA receptors: relevance to the ovarian cycle and stress. J. Neurosci. 27, 2155–2162. Maguire, J.L., Stell, B.M., Rafizadeh, M., Mody, I., 2005. Ovarian cycle-linked changes in GABAA receptors mediating tonic inhibition alter seizure susceptibility and anxiety. Nat. Neurosci. 8, 797–804. Matsui, D., Sakari, M., Sato, T., Murayama, A., Takada, I., Kim, M., Takeyama, K., Kato, S., 2002. Transcriptional regulation of the mouse steroid 5areductase type II gene by progesterone in brain. Nucleic Acids Res. 30, 1387–1393. McHugh, T.J., Jones, M.W., Quinn, J.J., Balthasar, N., Coppari, R., Elmquist, J.K., Lowell, B.B., Fanselow, M.S., Wilson, M.A., Tonegawa, S., 2007. Dentate gyrus NMDA receptors mediate rapid pattern separation in the hippocampal network. Science 317, 94–99. Milligan, C.J., Buckley, N.J., Garret, M., Deuchars, J., Deuchars, S.A., 2004. Evidence for inhibition mediated by coassembly of GABAA and GABAC receptor subunits in native central neurons. J. Neurosci. 24, 7241– 7250. Mitev, Y.A., Darwish, M., Wolf, S.S., Holsboer, F., Almeida, O.F., Patchev, V.K., 2003. Gender differences in the regulation of 3a-hydroxysteroid dehydrogenase in rat brain and sensitivity to neurosteroid-mediated stress protection. Neuroscience 120, 541–549. Mohler, H., 2007. Molecular regulation of cognitive functions and developmental plasticity: impact of GABAA receptors. J. Neurochem. 102, 1–12. Morris, K.D., Amin, J., 2004. Insight into the mechanism of action of neuroactive steroids. Mol. Pharmacol. 66, 56–69.
E.A. Mitchell et al. / Neurochemistry International 52 (2008) 588–595 Nusser, Z., Mody, I., 2002. Selective modulation of tonic and phasic inhibitions in dentate gyrus granule cells. J. Neurophysiol. 87, 2624–2628. Nusser, Z., Sieghart, W., Stephenson, F.A., Somogyi, P., 1996. The a6 subunit of the GABAA receptor is concentrated in both inhibitory and excitatory synapses on cerebellar granule cells. J. Neurosci. 16, 103–114. Nusser, Z., Sieghart, W., Somogyi, P., 1998. Segregation of different GABAA receptors to synaptic and extrasynaptic membranes of cerebellar granule cells. J. Neurosci. 18, 1693–1703. Peters, J.A., Kirkness, E.F., Callachan, H., Lambert, J.J., Turner, A.J., 1988. Modulation of the GABAA receptor by depressant barbiturates and pregnane steroids. Br. J. Pharmacol. 94, 1257–1269. Pinna, G., Agis-Balboa, R.C., Doueiri, M.S., Guidotti, A., Costa, E., 2004. Brain neurosteroids in gender-related aggression induced by social isolation. Crit. Rev. Neurobiol. 16, 75–82. Poisbeau, P., Patte-Mensah, C., Keller, A.F., Barrot, M., Breton, J.D., LuisDelgado, O.E., Freund-Mercier, M.J., Mensah-Nyagan, A.G., Schlichter, R., 2005. Inflammatory pain upregulates spinal inhibition via endogenous neurosteroid production. J. Neurosci. 25, 11768–11776. Porcello, D.M., Huntsman, M.M., Mihalek, R.M., Homanics, G.E., Huguenard, J.R., 2003. Intact synaptic GABA-ergic inhibition and altered neurosteroid modulation of thalamic relay neurons in mice lacking d subunit. J. Neurophysiol. 89, 1378–1386. Pritchett, D.B., Seeburg, P.H., 1990. g-aminobutyric acidA receptor a5-subunit creates novel type II benzodiazepine receptor pharmacology. J. Neurochem. 54, 1802–1804. Pritchett, D.B., Sontheimer, H., Shivers, B.D., Ymer, S., Kettenmann, H., Schofield, P.R., Seeburg, P.H., 1989. Importance of a novel GABAA receptor subunit for benzodiazepine pharmacology. Nature 338, 582–585. Reddy, D.S., 2003. Is there a physiological role for the neurosteroid THDOC in stress-sensitive conditions? Trends Pharmacol. Sci. 24, 103–106. Represa, A., Ben Ari, Y., 2005. Trophic actions of GABA on neuronal development. Trends Neurosci. 28, 278–283. Reynolds, D.S., Rosahl, T.W., Cirone, J., O’Meara, G.F., Haythornthwaite, A., Newman, R.J., Myers, J., Sur, C., Howell, O., Rutter, A.R., Atack, J., Macaulay, A.J., Hadingham, K.L., Hutson, P.H., Belelli, D., Lambert, J.J., Dawson, G.R., McKernan, R., Whiting, P.J., Wafford, K.A., 2003. Sedation and anesthesia mediated by distinct GABAA receptor isoforms. J. Neurosci. 23, 8608–8617. Rick, C.E., Ye, Q., Finn, S.E., Harrison, N.L., 1998. Neurosteroids act on the GABAA receptor at sites on the N-terminal side of the middle of TM2. Neuroreport 9, 379–383. Rudolph, U., Mohler, H., 2006. GABA-based therapeutic approaches: GABAA receptor subtype functions. Curr. Opin. Pharmacol. 6, 18–23. Saxena, N.C., Macdonald, R.L., 1994. Assembly of GABAA receptor subunits: role of the d subunit. J. Neurosci. 14, 7077–7086. Schweizer, C., Balsiger, S., Bluethmann, H., Mansuy, I.M., Fritschy, J.M., Mohler, H., Luscher, B., 2003. The g2 subunit of GABAA receptors is required for maintenance of receptors at mature synapses. Mol. Cell. Neurosci. 24, 442–450.
595
Semyanov, A., Walker, M.C., Kullmann, D.M., Silver, R.A., 2004. Tonically active GABAA receptors: modulating gain and maintaining the tone. Trends Neurosci. 27, 262–269. Shen, H., Gong, Q.H., Aoki, C., Yuan, M., Ruderman, Y., Dattilo, M., Williams, K., Smith, S.S., 2007. Reversal of neurosteroid effects at a4b2d GABAA receptors triggers anxiety at puberty. Nat. Neurosci. 10, 469–477. Sieghart, W., 1995. Structure and pharmacology of g-aminobutyric acidA receptor subtypes. Pharmacol. Rev. 47, 181–234. Sieghart, W., 2006. Structure, pharmacology, and function of GABAA receptor subtypes. Adv. Pharmacol. 54, 231–263. Sieghart, W., Sperk, G., 2002. Subunit composition, distribution and function of GABAA receptor subtypes. Curr. Top. Med. Chem. 2, 795–816. Sigel, E., Buhr, A., 1997. The benzodiazepine binding site of GABAA receptors. Trends Pharmacol. Sci. 18, 425–429. Stell, B.M., Brickley, S.G., Tang, C.Y., Farrant, M., Mody, I., 2003. Neuroactive steroids reduce neuronal excitability by selectively enhancing tonic inhibition mediated by delta subunit-containing GABAA receptors. Proc. Natl. Acad. Sci. U.S.A. 100, 14439–14444. Thomas, P., Mortensen, M., Hosie, A.M., Smart, T.G., 2005. Dynamic mobility of functional GABAA receptors at inhibitory synapses. Nat. Neurosci. 8, 889–897. Torres, J.M., Ortega, E., 2003. Differential regulation of steroid 5a-reductase isozymes expression by androgens in the adult rat brain. FASEB J. 17, 1428–1433. Ueno, S., Tsutsui, M., Toyohira, Y., Minami, K., Yanagihara, N., 2004. Sites of positive allosteric modulation by neurosteroids on ionotropic g-aminobutyric acid receptor subunits. FEBS Lett. 566, 213–217. Upasani, R.B., Yang, K.C., Acosta-Burruel, M., Konkoy, C.S., McLellan, J.A., Woodward, R.M., Lan, N.C., Carter, R.B., Hawkinson, J.E., 1997. 3aHydroxy-3b-(phenylethynyl)-5b-pregnan-20-ones: synthesis and pharmacological activity of neuroactive steroids with high affinity for GABAA receptors. J. Med. Chem. 40, 73–84. van Rijnsoever, C., Tauber, M., Choulli, M.K., Keist, R., Rudolph, U., Mohler, H., Fritschy, J.M., Crestani, F., 2004. Requirement of a5-GABAA receptors for the development of tolerance to the sedative action of diazepam in mice. J. Neurosci. 24, 6785–6790. Weir, C.J., Ling, A.T., Belelli, D., Wildsmith, J.A., Peters, J.A., Lambert, J.J., 2004. The interaction of anaesthetic steroids with recombinant glycine and GABAA receptors. Br. J. Anaesth. 92, 704–711. Wittmer, L.L., Hu, Y., Kalkbrenner, M., Evers, A.S., Zorumski, C.F., Covey, D.F., 1996. Enantioselectivity of steroid-induced g-aminobutyric acidA receptor modulation and anesthesia. Mol. Pharmacol. 50, 1581–1586. Wohlfarth, K.M., Bianchi, M.T., Macdonald, R.L., 2002. Enhanced neurosteroid potentiation of ternary GABAA receptors containing the d subunit. J. Neurosci. 22, 1541–1549. Wulff, P., Goetz, T., Leppa, E., Linden, A.M., Renzi, M., Swinny, J.D., Vekovischeva, O.Y., Sieghart, W., Somogyi, P., Korpi, E.R., Farrant, M., Wisden, W., 2007. From synapse to behavior: rapid modulation of defined neuronal types with engineered GABAA receptors. Nat. Neurosci. 10, 923–929.