CHAPTER SIX

Inhibitory Neurosteroids and the GABAA Receptor Sandra Seljeset, Duncan Laverty, Trevor G. Smart1 Department of Neuroscience, Physiology and Pharmacology, UCL, London, United Kingdom 1 Corresponding author: e-mail address: [email protected]

Contents 1. 2. 3. 4.

Introduction Structure–Function of Inhibitory Neurosteroids Physiological Effects of Inhibitory Neurosteroids at GABAARs Potential Inhibitory Neurosteroid-Binding Sites on GABAARs 4.1 The GABAAR ion channel at the 20 position 5. The Potentiating Neurosteroid-Binding Site Is Unaffected by Inhibitory Neurosteroids 6. Inhibitory Neurosteroid-Binding Site Outside the Ion Channel—C. elegans and UNC-49 7. Conclusion Conflict of Interest Acknowledgment References

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Abstract γ-Aminobutyric acid type A receptors (GABAARs) are vital proteins that are engaged in regulating neural circuit activity in the central nervous system. Their effectiveness in this task is dependent on the extent of receptor modulation by naturally occurring ligands that are released in the brain. One of the foremost examples of such ligands is the neurosteroids that can either potentiate GABAAR function or cause direct inhibition. To fully understand the underlying mechanisms by which neurosteroids modulate GABAARs, it is necessary to identify their binding sites on the receptors. For potentiating neurosteroids, recent work has made substantive progress in identifying a binding site located in the transmembrane domains of GABAAR α subunits. However, for the inhibitory neurosteroids, several possibilities exist including an ion channel site as well as potential sites in the transmembrane domain. This review systematically analyzes the evidence behind possible binding sites for the inhibitory neurosteroids. We consider the chemical structure–function properties of such inhibitory neurosteroids, their physiological effects on synaptic inhibition, and whether a binding site exists in the GABA ion channel or in other areas of the transmembrane domain. Finally, we discuss how structural homology modeling and Cys-loop receptor homologues may help to locate the inhibitory neurosteroid-binding site on GABAARs. Advances in Pharmacology, Volume 72 ISSN 1054-3589 http://dx.doi.org/10.1016/bs.apha.2014.10.006

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ABBREVIATIONS ALLOP allopregnanolone CNS central nervous system DHEAS dehydroepiandrosterone sulfate Ent enantiomer GAT GABA transporters P peak PS pregnenolone sulfate SS steady state TBPS t-butyl-bicyclophosphoro-thionate THDOC tetrahydro-deoxycorticosterone

1. INTRODUCTION γ-Aminobutyric acid type A receptors (GABAARs) are important membrane proteins in the central nervous system (CNS) for neural development and for ensuring that innate neuronal excitability is controlled (Ben Ari, Gaiarsa, Tyzio, & Khazipov, 2007; Fritschy & Panzanelli, 2014). Controlling neuronal excitability relies on two principal forms of inhibition: a transient activation of synaptic GABAARs (phasic) and a persistent, but less intense activation of extrasynaptic receptors (tonic; Farrant & Nusser, 2005; Mody, 2001; Semyanov, Walker, Kullmann, & Silver, 2004). Both these forms of inhibition are present in most regions of the CNS. The importance of these roles for GABAARs becomes evident when either GABA release or receptor function and/or trafficking becomes dysfunctional, often resulting in a variety of neurological disorders (Hines, Davies, Moss, & Maguire, 2012; Smith & Rudolph, 2012). The efficiency with which GABA performs these inhibitory “housekeeping” roles will depend upon numerous factors, including on the postsynaptic side: the number and location of GABAARs on neurons and the subtypes of GABAAR expressed in individual neurons (Fritschy & Brunig, 2003; Nusser, Hajos, Somogyi, & Mody, 1998). Presynaptically, the extent of GABA release will also be important, which in turn may be affected by the activity of presynaptic GABAA (Cossart, Bernard, & Ben Ari, 2005; Gaiarsa, Caillard, & Ben Ari, 2002; Ruiz, Campanac, Scott, Rusakov, & Kullmann, 2010), endocannabinoid (Katona & Freund, 2008), and NMDA receptors (Duguid, 2012; Duguid & Smart, 2009), as well as many other ligand-gated and G-protein-coupled receptors (Khakh & Henderson, 2000). Over time, the steady-state (SS) levels of

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GABA in and around inhibitory synapses will also be subject to clearance by neighboring glial- and neuronal-based GABA transporters (GAT1–3; Borden, 1996). The extent of GABA inhibition can additionally be affected by a raft of extracellular and intracellular endogenous modulators such as protons (Huang & Dillon, 1999; Krishek, Amato, Connolly, Moss, & Smart, 1996; Wilkins, Hosie, & Smart, 2002, 2005), Zn2+ (Harrison & Gibbons, 1994; Hosie, Dunne, Harvey, & Smart, 2003; Smart, Xie, & Krishek, 1994); protein kinases (Brandon, Jovanovic, & Moss, 2002; Kittler & Moss, 2003; Luscher, Fuchs, & Kilpatrick, 2011), and the neurosteroids (Belelli et al., 2006; Belelli & Lambert, 2005)—all of which alone, or in combination, can regulate synaptic and extrasynaptic GABAAR activities. The neurosteroids are of particular interest since they form a group of potent modulators for GABAARs, exhibiting a range of effects from potentiation of GABA responses and direct receptor activation, to inhibition (Belelli & Lambert, 2005). Neurosteroids that potentiate GABA activity, but which under specific conditions can also directly activate the receptor, are referred to as “potentiating neurosteroids,” while those that antagonize are termed “inhibitory neurosteroids.” Generically, neurosteroids are synthetically derived in both neurons and glia from cholesterol, via pregnenolone, a major precursor for the neuroactive steroids (Compagnone & Mellon, 2000). The activity of 3β-dehydrogenase converts pregnenolone into the sex hormone progesterone, which is converted into a major GABA-potentiating neurosteroid, allopregnanolone (5α-pregan-3α-ol-20-one; ALLOP) by a 5α-reductase. Progesterone is also a key intermediate for generating (via a 21β-hydroxylase) the stress hormone, deoxycorticosterone. This is subsequently converted into tetrahydro-deoxycorticosterone (5α-pregnan-3α,21diol-20-one; THDOC; Belelli & Lambert, 2005; Lambert, Belelli, Peden, Vardy, & Peters, 2003), another GABA-potentiating neurosteroid. Thus, progesterone and deoxycorticosterone are major precursors for the potentiating neurosteroids (Rupprecht & Holsboer, 1999; Tsutsui, 2006). The inhibitory neurosteroids incorporate a subclass known as the sulfated steroids (Gibbs, Russek, & Farb, 2006), though sulfation is not obligatory for inhibition. This family of neurosteroids is naturally occurring in the CNS and can antagonize GABAAR function (Majewska & Schwartz, 1987; Wang et al., 2002). Pregnenolone is the main precursor of pregnenolone sulfate (PS) and dehydroepiandrosterone sulfate (DHEAS), which are formed by the action of hydroxylases and hydroxysulfotransferases (Tsutsui, 2006; Wang, 2011).

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The potent interaction that neurosteroids have with GABAARs translates into numerous physiological effects within the CNS. Potentiating neurosteroids, at low nanomolar concentrations, coassociate with stress (Purdy, Morrow, Moore, & Paul, 1991), alcohol intoxication (Kumar, Fleming, & Morrow, 2004), and pregnancy/estrus (Concas, Follesa, Barbaccia, Purdy, & Biggio, 1999) and possibly involve the potentiation of GABA responses (Belelli & Herd, 2003; Stell, Brickley, Tang, Farrant, & Mody, 2003; Zhu & Vicini, 1997). Neurosteroids have also been linked to anxiolysis, antidepression, sedation/hypnosis, anticonvulsion, and anesthesia (Barbaccia, 2004; Bitran, Shiekh, & McLeod, 1995), with impaired production associated with premenstrual dysphoric disorder (Backstrom et al., 2003; Maguire, Stell, Rafizadeh, & Mody, 2005), panic disorder (Brambilla et al., 2003; Eser et al., 2006), depression (Uzunova, Sampson, & Uzunov, 2006), schizophrenia, and bipolar disorder (Marx et al., 2006). Less is known about the physiological effects of the inhibitory neurosteroids though PS is associated with cognitive antiamnesic effects (Ladurelle et al., 2000; Vallee et al., 2001) and raised levels of dehydroepiandrosterone in the hippocampus have been noted in Alzheimer’s disease (Brown, Han, Cascio, & Papadopoulos, 2003), which might be relevant during aging. Furthermore, PS has biphasic effects regarding absence of epilepsy (Citraro et al., 2006) and can exhibit proconvulsant (Reddy & Kulkarni, 1998) and convulsant properties (Williamson, Mtchedlishvili, & Kapur, 2004). To probe the mechanisms by which neurosteroids affect GABAAR function, it is helpful to identify their binding sites. However, this task has not been straightforward. Essentially, the potentiating steroids most likely occupy a transmembrane domain site within receptor α subunits (Akk et al., 2007; Hosie, Clarke, da Silva, & Smart, 2009; Hosie, Wilkins, da Silva, & Smart, 2006; Hosie, Wilkins, & Smart, 2007). However, the precise molecular identification of an inhibitory neurosteroid-binding site has been more problematic to solve, though it is probably independent from that of the potentiating site. Thus, the aim of this review is to appraise current ideas as to where the inhibitory neurosteroids are most likely to be binding at the GABAAR. To do this, we will address three important areas with regard to inhibitory neurosteroids and GABAARs: their structure–function properties, their physiological effects on neuronal activity, and studies on the molecular dissection of likely neurosteroid-binding sites.

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2. STRUCTURE–FUNCTION OF INHIBITORY NEUROSTEROIDS The important molecular determinants that mediate neurosteroidinduced inhibition at GABAARs are not yet fully understood. Sulfated steroids such as PS (Fig. 1A) and DHEAS (Fig. 1B) are naturally produced and released in the brain. They are less potent than the potentiating steroids and seemingly act as noncompetitive antagonists at GABAARs (Akk, Bracamontes, & Steinbach, 2001; Majewska, Demirgoren, Spivak, & London, 1990; Majewska, Mienville, & Vicini, 1988; Woodward, Polenzani, & Miledi, 1992) by apparently binding to a discrete site from that for the potentiating neurosteroids ALLOP and THDOC (Akk et al., 2008; Majewska, Demirgoren, & London, 1990; Park-Chung, Malayev, Purdy, Gibbs, & Farb, 1999). The inhibitory neurosteroids clearly differ from the potentiating neurosteroids in their actions at GABAARs. This is probably reflected, at least in part, by their structural diversity. Inhibitory steroids demonstrate flexibility in the permitted molecular structure and conformation that preserves inhibition. For example, we know that the conformation of the substituent at position C3 in the A ring (Fig. 1A) is not critical for inhibition, as 3α (below the plane of the A ring) and 3β (above plane) PS both exhibit profound inhibition of GABA currents. In addition, the conformation at C5 is not critical either, since 5β-pregnan-3β-ol-20-one-sulfate and 5α-pregnan-3βol-20-one-sulfate are equally effective as inhibitors (Fig. 1C and D) and they also have similar efficacy to PS (5β-pregnen-3β-ol-20-one-sulfate), which possesses a double bond at C5–C6 (Fig. 1A; Park-Chung et al., 1999). However, inhibition is dependent on position C11 in the C ring, since 11β-hydroxy-PS retains inhibitory activity but this changes to potentiation following substitution for a ketone (11-keto-pregnenolone sulfate; Fig. 1F). This switch in effect does not occur if the keto group is relocated to C7 (Park-Chung et al., 1999). In ring D, the nature of the group at C17, with regard to inhibition, is less stringent given that acetyl, acetoxy, and keto groups substituted onto a PS background retain similar inhibitory activities. The chemical nature of the substituent at C3 is similarly less stringent with the much larger hemisuccinate group or a smaller hydroxyl group both capable of supporting inhibition by neurosteroids, but the relative efficacy is substantially reduced when compared with C3 linked to a sulfate group in

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Dehydro-epiandrosterone sulfate (DHEAS)

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Pregnan

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11β-Hydroxy-pregnenolone sulfate G

11-Keto-pregnenolone sulfate H

17β-Estradiol-3β-sulfate I

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Figure 1 Chemical structures of naturally occurring and synthetic inhibitory neurosteroids. Ring labels and numbering are shown for pregnenolone sulfate and applies to all other structures. The chiral centers for pregnenolone sulfate and for DHEAS are identified by circles. Ent—enantiomer.

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PS. Apart from molecular volume, the hemisuccinate group is charged and the hydroxyl will only develop a dipole compared to the charged sulfate group. Thus, for inhibition, it is not an absolute requirement to have a charged group at C3 (Wang et al., 2002). Despite the conformational and structural flexibility at C3, a completely unsaturated ring A does reduce inhibition (Fig. 1G) even if the C3 substituent is a sulfate or a much larger benzoate group (Park-Chung et al., 1999). In conclusion, although many areas of inhibitory steroid chemical structure remain equivocal, we can state that for steroids to exhibit efficacious inhibitory activity, ring A should be saturated and position C3 should retain a sulfate group, with negative charge increasing inhibition, as exemplified by comparing the markedly potentiating androsterone (Fig. 1H), with its inhibitory, sulfate-containing, counterpart, androsterone sulfate (Fig. 1I). Furthermore, if a hemisuccinate group is preferred at C3, then C5–C6 in ring B should be saturated. Whether this requirement is also pertinent to other substituents at C3 (e.g., hydroxyl) remains to be determined. The stereochemistry at C3 is relatively less important for inhibitory steroids, contrasting with the strict requirement for a 3α conformation for potentiating steroids. The stereochemistry at C5 is also largely irrelevant, and many different substituents placed at C17 are broadly tolerated in terms of inhibition. However, a difference in inhibitory potency has been reported for some enantiomers of sulfated steroids (Fig. 1J). For DHEAS, the naturally occurring and corresponding enantiomer differ by sevenfold in potency for inhibiting GABA-activated currents, while no differences were resolved for PS and its corresponding enantiomer in rat hippocampal neurons (Nilsson, Zorumski, & Covey, 1998), which may indicate that their inhibitory effects are mediated via different binding sites. Surprisingly, the reverse result was obtained for a Caenorhabditis elegans mutant UNC-49B GABA receptor. In this instance, PS does exhibit enantiomeric selectivity, with the natural enantiomer being threefold more potent than its synthetic counterpart, while DHEAS showed no enantiomeric selectivity (Twede, Tartaglia, Covey, & Bamber, 2007). Enantiomeric selectivity is often used as evidence for a specific ligand-binding site on a protein, and when this is lacking, indirect interactions between the ligand and the membrane are considered more likely. However, this simple “rule-of-thumb” may not be applicable to neurosteroid binding and the mammalian GABAA and C. elegans GABA receptor may be showing genuinely subtle differences in the their binding sites for the inhibitory neurosteroids, which we explore in the last section of this review.

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3. PHYSIOLOGICAL EFFECTS OF INHIBITORY NEUROSTEROIDS AT GABAARs The type of inhibition of GABAAR function caused by inhibitory neurosteroids has been subjected to considerable study and provides useful information in probing for potential binding sites for these steroids on GABA receptors. Current evidence points toward two potential binding site locations, one quite specific, within the GABA ion channel, and the other site located in an as-yet undetermined position outside the ion channel domain. The evidence for these binding site locations originates from studies using both recombinant and native GABAARs. At recombinant α1β2γ2L receptors, PS is a more potent blocker when used with higher concentrations of GABA (Eisenman, He, Fields, Zorumski, & Mennerick, 2003), an observation usually indicative of a binding site that is more easily accessible when the receptor is activated (use dependence). Receptor activation seemed more important than agonist occupancy of the neurotransmitter-binding site since comparing GABA with a partial agonist revealed that the relative maximal efficacy rather than the agonist EC50 affected the extent of the block (Eisenman et al., 2003). Indeed, by matching fractional agonist responses (similar open probability) to both full and partial agonists, PS was equally efficacious as a blocker. The charged nature of the sulfate group on PS, and its ability to increase inhibition, might suggest a voltage-sensitive mechanism of block, particularly if this group is exposed to the membrane electric field by penetrating into the GABA ion channel. However, PS displays very weak voltage sensitivity, though a C3-carboxylated pregnane steroid derivative (3α,5β-20oxo-pregnane-3-carboxylic acid) does exhibit voltage-sensitive inhibition at depolarized membrane potentials. The actions of this steroid are complex, featuring GABA potentiation, and at high concentrations, overt inhibitory effects (Covey, Evers, Mennerick, Zorumski, & Purdy, 2001; Mennerick et al., 2001). The voltage-sensitive inhibition was independent of carboxylate stereochemistry at C3, and switching the location of this group from ring A to ring D at C24 curiously did not affect the inhibition (Mennerick et al., 2001), suggesting a flexible, accommodating, or perhaps only partially occupied binding site. Given the likely dissociated state of the carboxyl group at physiological pH, decreasing external pH relieves inhibition, probably due to reduced ionization of the carboxylated steroid. Whether the potentiating and inhibitory effects are mediated by one or more binding sites is unclear.

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Nevertheless, for inhibition, it should be noted that the appearance of voltage sensitivity need not necessarily invoke the presence of an ion channelbinding site. Deducing whether inhibition by PS is affected by the activation state of the GABAAR is complicated. Coapplication of GABA and PS revealed a greater block of the SS current when compared to the peak (P) current, which may reflect a slow on-binding rate for PS, or a state-dependent block of the receptor that requires time to develop. For example, it could reflect the entry of the receptor into one or more desensitized states that are more sensitive to block by PS. However, prolonged coapplication of PS did not increase the SS block suggesting that receptor activation by GABA was more important for establishing inhibition (Eisenman et al., 2003). Receptor deactivation following removal of GABA from the receptor after a prolonged application was delayed by PS, contrasting with the faster deactivation rates after only brief GABA applications (Eisenman et al., 2003; Shen, Mennerick, Covey, & Zorumski, 2000), reinforcing the view of a statedependent interaction of the inhibitor with GABAARs. The slow rate of onset for inhibition by PS was increased by raising the blocker concentration, but was independent of GABA concentration used to activate the receptor. However, increasing GABA concentration slowed the rate of recovery from PS block, accentuating the level of block at higher GABA concentrations. This result was seemingly not in accord with a channel-blocking site, or even a “trapped-blocker”-binding site, that can be relieved by channel opening allowing blocker dissociation. This also differs from the block produced by ligands with assumed channel-binding sites at GABAARs such as picrotoxin and similar antagonists (e.g., t-butyl-bicyclophosphoro-thionate (TBPS)), which is relieved by high concentrations of GABA (Bali & Akabas, 2007; Smart & Constanti, 1986; Van et al., 1987; Yoon, Covey, & Rothman, 1993). At the single GABA channel level, analysis of clusters of GABA channel activity induced by low and high GABA concentrations suggested that while PS reduced cluster duration, both the shut and open states of GABA channels seemed equally susceptible to block (Akk et al., 2001). Furthermore, GABA channel opening and closing rates were unaffected by PS. The mixed and complicated kinetic profiles about the nature of the PS block preclude the generation of a single unifying mechanism that can accommodate all of the actions of inhibitory steroids. That said, many effects can be explained by this particular blocker binding to possibly many activated states of the GABAAR and promoting its residency in a desensitized state (Eisenman et al., 2003; Shen et al., 2000; Wang et al., 2002).

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4. POTENTIAL INHIBITORY NEUROSTEROID-BINDING SITES ON GABAARs 4.1. The GABAAR ion channel at the 20 position Given the noncompetitive nature of PS inhibition and its similarity, in part, to the mechanism of block of GABAARs by picrotoxin, the GABA ion channel is considered to be a potential location for the inhibitory neurosteroid-binding site. This notion was reinforced by early studies reporting that picrotoxin can displace PS in rat brain membranes (Majewska, Demirgoren, & London, 1990) and that PS competitively inhibits the binding of the picrotoxin-like blocker and convulsant, TBPS. However, the sulfated inhibitory neurosteroid, DHEAS, had no effect on TBPS binding (Majewska & Schwartz, 1987). Although radioligand displacement is often used as an indicator of competition for the same binding site, for allosteric proteins, binding displacement need not imply a common binding site. One of the earliest studies searching for the inhibitory neurosteroidbinding site mutated two threonine residues (T271F and T277A at 60 and 120 , respectively) in the γ2 subunit ion channel lining that rendered α1β2γ2 receptors insensitive to picrotoxin (Gurley, Amin, Ross, Weiss, & White, 1995). However, antagonism by PS and by DHEAS was unaffected, suggesting that the determinants of their inhibitory action are not shared with picrotoxin (Shen, Mennerick, Zorumski, Covey, & Zorumski, 1999). Other studies of picrotoxin inhibition at GABAARs mutated residues at the 20 position in the M2 ion channel lining for receptor α and/or β subunits, rather than the γ2 subunit (Ffrench-Constant, Rocheleau, Steichen, & Chalmers, 1993; Xu, Covey, & Akabas, 1995; Zhang, Ffrench-Constant, & Jackson, 1994). This location for the picrotoxin-binding site (Figs. 2 and 3) is supported by the crystal structure for the Cys-loop receptor homolog, glutamate-activated Cl channel (GluCl) showing picrotoxin in situ in the ion channel (Hibbs & Gouaux, 2011). Moreover, a homologous residue also reduces the block by picrotoxin of GABAA ρ1 receptors (Wang, Hackam, Guggino, & Cutting, 1995). When the 20 position (valine 256; Fig. 2A and C) in α1 subunits (α1V256) was mutated to serine and expressed in HEK cells as α1V256Sβ2γ2L receptors, the apparent association rate for PS was reduced by approximately 30-fold, suggesting that the ability of PS to block these receptors is reduced by including serine at the 20 position in the α1 subunit (Akk et al., 2001). Single-channel

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Figure 2 GABAA receptor homology models based on templates using the glutamateactivated Cl channel from C. elegans (PDB 3rhw). The schematic shows a plan view of the GABAA receptor with the extracellular domain removed revealing the transmembrane domains and ion channel lining formed by M2 for α1, β2, and γ2 subunits. The wild-type receptor (A) shows the residues at the 20 position around a presumed picrotoxin-binding site. The mutant receptor contains the substitution α1V256S (B). (C) Side view for the wild-type GABA ion channel at the 20 position showing the respective residues for the two α subunits (V256) and one β subunit (A252). (D) A similar view at the 20 position for the mutant GABAA receptor with α1V256S.

cluster durations in the presence of PS were no longer reduced. Furthermore, mutating the corresponding 20 positions in the β2 (A252S) or γ2 (S266A; Fig. 3) subunits did not affect the PS block. Despite these findings, other studies measuring whole-cell GABA currents report that the inhibitory effect of PS is reduced or even abolished in Xenopus oocytes expressing α1β2A252Sγ2L or α1V256Sβ2γ2L (Wang, Rahman, Zhu, & Backstrom, 2006; Wang, Rahman, Zhu, Johansson, & Backstrom, 2007), indicating that the 20 mutation in either α1 or β2 subunits can abolish sensitivity to PS. We should note that a single point mutation by itself does not identify a ligand-binding site without corroborating evidence. We know that the block by PS is dependent upon receptor activation (Eisenman et al., 2003) and that it is also independent of the membrane

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A

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Figure 3 Schematic diagram of the transmembrane topology for a GABA receptor subunit (A) and also presented in a linear format (B). (C) Primary sequence alignment for GABAA receptor α1, β3, γ2, and ρ1 subunits in comparison with homologous regions for the UNC-49B and UNC-49C C. elegans GABA receptor subunits. The transmembrane domains (M1–M3) are boxed, note only part of M3 is shown. The prime numbering notation for M2 is shown. The M1–M2 and M2–M3 linkers are also included. Residues referred to in the text are boxed and highlighted. See the online version for color (different gray shades in print) coding of individual residues used to identify areas of homology or clear divergence among the subunit sequences.

potential (Akk et al., 2001; Eisenman et al., 2003), suggesting that the negatively charged sulfate group does not move through the membrane electric field. If the 20 residue was involved in PS binding, a greater level of block would be expected at more depolarized membrane potentials. Therefore, these observations could imply that the 20 residue is mostly involved in the signal transduction mechanism following binding by PS to another site and that the mutation at 20 simply alters an allosteric mechanism to initiate inhibition. The potentiating neurosteroids (THDOC, ALLOP) are unaffected by the 20 mutation, suggesting that inhibitory and potentiating neurosteroids are using different signal transduction pathways (Wang et al., 2002) and most likely different binding sites. Another group of PS-like GABA antagonists are the 3βhydroxypregnane steroids (Wang et al., 2002). These compounds are diastereomers of the potentiating 3α-hydroxypregnane steroids, but are similar to the sulfated neurosteroids in that they cause noncompetitive inhibition at the GABAAR in an activation- or state-dependent manner. Interestingly, the 20 mutation (α1V256S) also eliminated GABAAR antagonism by the

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3β-hydroxypregnane steroids, but they were unaffected by the equivalent β2 subunit 20 mutation (A252S; Wang et al., 2007) suggesting a difference in the mechanism of block compared to PS. In this latter study, desensitization was quantified by determining a P to SS current ratio (P/SS). In wild-type receptors, PS increased this ratio in a dose-dependent manner, whereas for the α1 and β2 ion channel mutants, the ratio remained unaffected by changes in concentration. This could be interpreted as promotion into the desensitized state by PS at wild-type receptors, and possibly stabilization of the receptor in one or more desensitized states. This effect is removed by the 20 mutations. By contrast, inhibition by the 3β-hydroxypregnane steroid does not cause a dose-dependent increase of the P/SS ratio in wild-type or mutant receptors, suggesting that the mechanism of block by sulfated steroids and 3β-hydroxypregnane steroids is indeed different. It also implies that the 20 residue is unlikely to be the binding site for either group of steroids, but rather that it has a role in the signal transduction of the inhibitory effect.

5. THE POTENTIATING NEUROSTEROID-BINDING SITE IS UNAFFECTED BY INHIBITORY NEUROSTEROIDS For the potentiating neurosteroids, a binding site has been located in the transmembrane region of α1 subunits involving Q241 in M1, and N407 and Y410 in M4 (Hosie et al., 2006, 2007). The principal residue is Q241 (Fig. 3), which is located at the base of a water-filled cavity between the M1– M4 interface of the α1 subunit and is conserved among all members of the α subunit family (α1–6; Hosie et al., 2009). This aqueous cavity surrounded by M1–M4 is thought to increase in depth and volume upon receptor activation ( Jung, Akabas, & Harris, 2005; Lobo, Mascia, Trudell, & Harris, 2004; Williams & Akabas, 1999), allowing potentiating neurosteroids to bind to Q241 and stabilize the activated receptor complex in an open conformation. The sulfated neurosteroid, DHEAS, does not compete with 3αhydroxy-5β-pregnan-20-one (pregnanolone) for binding at recombinant α1β2γ2S receptors, suggesting that the inhibitory and potentiating neurosteroids do not share a binding site (Park-Chung et al., 1999). Furthermore, the mutation α1Q241W does not affect inhibition by PS (Akk et al., 2008), whereas it abolished the effects of the potentiating neurosteroids (Hosie et al., 2006). Hence, it is unlikely that potentiating and inhibitory neurosteroids share a binding site, and the site for inhibition by neurosteroids is likely to be located elsewhere on the GABAAR.

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6. INHIBITORY NEUROSTEROID-BINDING SITE OUTSIDE THE ION CHANNEL—C. ELEGANS AND UNC-49 The Cl-permeable C. elegans GABA receptor, UNC-49, is encoded by one gene that generates three alternatively spliced variants containing a shared N-terminus with three different C termini: UNC-49A, UNC49B, and UNC-49C (Bamber, Beg, Twyman, & Jorgensen, 1999; Bamber, Twyman, & Jorgensen, 2003). These are structurally and pharmacologically closely related to the mammalian GABAAR, possessing an external N-terminal domain and four transmembrane domains (Fig. 3). Whereas UNC-49A is expressed at low levels, higher levels of UNC-49B and UNC49C are found at the neuromuscular junction of C. elegans (Bamber et al., 1999; Bamber, Richmond, Otto, & Jorgensen, 2005). UNC-49B will assemble as a pentameric homomer in vitro and in vivo, whereas UNC49C can only form functional receptors at synapses in vivo following coassembly with UNC-49B (Bamber et al., 2005). PS is approximately 80-fold more potent as an inhibitor at UNC-49B/C heteromers compared to UNC-49B homomers (with IC50s of 2.3 μM and approximately 191 μM, respectively), suggesting that the UNC-49C subunit contains residues that are important for PS inhibition (Wardell et al., 2006). This disparity in potency between the UNC receptors has enabled the determinants of PS inhibition to be identified, by using chimeras, and by substituting residues between UNC-49C and UNC-49B, eventually conferring complete sensitivity to PS on UNC-49B. Chimeras formed between UNC-49B and UNC-49C implied a key role for M1 and the extracellular M2–M3 linker of UNC-49C in PS inhibition (Wardell et al., 2006). Together, these regions fully accounted for the sensitivity to PS. Specific residues within these domains were then identified that are conserved among neurosteroid-sensitive receptors but lacking in UNC49B. The M2–M3 linker in UNC-49B was an obvious target due to notable differences in the primary sequence, especially residues with neutral and charged side chains compared to UNC-49C (Fig. 3). By creating an UNC-49B “background” chimera that contained M1 of UNC-49C, the neutral residues in the M2–M3 linker were serially examined. Mutating just one neutral asparagine (N305, original numbering includes the signal peptide) in the M2–M3 linker (top of M2; Fig. 4) to a positively charged arginine (N305R) found in UNC-49C conferred a substantive sensitivity to inhibition by PS inhibition (Wardell et al., 2006). We should note that more recent

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M4

Figure 4 GABAA receptor homology models for the C. elegans UNC-49B receptor, based on the crystal structure of the β3 GABA receptor homomer (PDB 4cof), for wild-type subunits (A) and one containing mutations discussed in the text (B). The extracellular domains are removed showing the tilted transmembrane domains and ion channel lining formed by M2. Those residues in M1 and the M2–M3 linker that affect pregnenolone sulfate- and DHEAS-induced inhibition are labeled.

homology models and a recent crystal structure of GABA β3 subunits (Miller & Aricescu, 2014) suggest that this residue is more likely to be at top of M2 rather than in the M2–M3 linker. Nothing more was gained by switching the entire M2–M3 linkers; thus, this one asparagine is sufficient to account for the increased sensitivity to PS that this linker confers to UNC-49B. A similar homology comparison of M1 between UNC-49B, UNC49C, and selected GABAAR subunits identified two more residues lacking in UNC-49B that are conserved among PS-sensitive receptors. These are an asparagine (N259) and a hydrophobic valine (V261) residues in UNC-49B that are replaced by glutamine (Q, in UNC-49C and selected GABAAR subunits) and by an aromatic residue (either phenylalanine (F, UNC49C) or tyrosine (Y, GABAARs), Figs. 3 and 4). However, UNC-49B subunits containing N259Q and V261F, in addition to the M2–M3 linker substitution, N305R, were only twofold more sensitive to PS than UNC-49B containing just the mutation, N305R (Fig. 4). This was sevenfold less sensitive than the chimera containing the whole of UNC-49C M1 and the M2–M3 linker. In addition, mutating F261 (UNC-49C) to valine (UNC-49B) did not reduce the

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sensitivity of UNC-49C to PS, suggesting that this residue is not essential for inhibition. Consequently, these results imply that other residues in UNC49C, besides N305, are probably involved in the binding of PS. A further five residues in M1 of UNC-49C were identified and, when substituted for those in UNC-49B, increased the sensitivity to pregnenolone sulfate: T257F, M258L, I262F, S264A, and I265S (Wardell et al., 2006). Methionine 258 is notable since its substitution with leucine in UNC-49B bestowed a biphasic response to PS, causing potentiation at 10 μM, but inhibition at 100 μM. Importantly, the specificity of all these mutations for inhibitory neurosteroids was emphasized by their lack of effect on picrotoxin sensitivity. The UNC-49C receptor is also sensitive to DHEAS. The comparable types of inhibition caused by DHEAS and PS suggested that these inhibitors may share a similar binding site(s) at the UNC-49C receptor. Indeed, most of the mutations previously observed (Wardell et al., 2006) to affect PS potency caused parallel changes to DHEAS potency, in accord with binding to the same region of the receptor. As for PS, DHEAS exhibits very little inhibitory activity at UNC-49B homomers, whereas UNC-49B/C heteromers are potently inhibited, suggesting that the major elements of the binding site are located within the UNC-49C subunit (Twede et al., 2007). Introducing the charged residue (N305R) into the M2–M3 linker increased the sensitivity of UNC-49B to higher concentrations of DHEAS (30 and 100 μM), while lower concentrations remained ineffective. By additionally incorporating M1 from UNC49C into the UNC-49B subunit, the sensitivity to DHEAS was increased to levels equivalent to that observed with UNC-49B/C heteromers. Although a number of substitutions in M1 were found to be necessary to increase the sensitivity to PS at UNC-49B (Wardell et al., 2006), to cause a similar increase in sensitivity to DHEAS required only the N259Q and V261F mutations in M1 in combination with the N305R in the M2–M3 linker (Twede et al., 2007; Figs. 3 and 4). Although DHEAS is equally potent as an inhibitor at UNC-49B when these three point mutations are incorporated, PS is sevenfold more potent at the chimera that contains the whole M1 helix of UNC-49C compared to the UNC-49B homomer with the three point mutations (Wardell et al., 2006), suggesting that there are differences between the molecular determinants coordinating the binding of PS and DHEAS. Differences in the molecular determinants were also apparent when considering specific point mutations. We have already noted that M258L

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confers a biphasic response on the UNC-49 receptor toward PS (potentiation at concentrations up to 30 μM and inhibition apparent at higher concentrations; Wardell et al., 2006); however, the same mutation caused just a small increase in DHEAS potency (Twede et al., 2007). Furthermore, S264A reduced DHEAS potency by two- to fivefold without affecting PS sensitivity. Overall, while these studies reveal that PS and DHEAS have a similar mechanism of action at the UNC-49B/C receptor, substituting the acetyl group at C17 for a carbonyl in DHEAS seems to affect how these inhibitory neurosteroids interact with the M1 helix. Given that the M1 domain of UNC-49C confers a sensitivity to PS and DHEAS onto UNC-49B (Twede et al., 2007; Wardell et al., 2006), the question arose as to whether the equivalent region in mammalian GABAARs was similarly influential toward the inhibitory neurosteroids. However, incorporating the M1 domain of UNC-49B into mammalian α1, β2, and γ2 subunits, to determine whether this removes the sensitivity to sulfated neurosteroids, was ineffective (Baker, Sturt, & Bamber, 2010). This suggested that the residues identified in UNC-49C as important for modulation by sulfated neurosteroids are unlikely to form a binding site that is conserved among different species.

7. CONCLUSION It is clear from the evidence presented here that the location for the binding site for inhibitory neurosteroids on GABAARs remains obscure, complex, and elusive. It seems that the location is very different from that for the potentiating neurosteroids, with the likeliest candidate identified to date being the ion channel lining, around the 20 residue, deep within the GABA ion channel and beyond the presumed location for the ion channel gate. However, the lack of voltage sensitivity for the sulfated steroids is confounding, and this argues for the 20 residue fulfilling a signaling role for inhibition rather than a direct binding role for the inhibitory neurosteroids. The alternative scenario involving M1 and residues at the top of M2 seem plausible to explain binding at the C. elegans GABA receptor, but the equivalent domain in mammalian GABAARs seems less certain as a binding locus for these ligands, despite elegant experiments in dissecting the functional domains of this receptor. Only by increasing our repertoire of tools and reagents will the inhibitory neurosteroid binding site become amenable

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to further molecular investigation. This is an important goal as this class of endogenous modulators of inhibitory neurotransmitter receptor function may be physiologically beneficial, not least given their impact on memory and cognition.

CONFLICT OF INTEREST The authors have no conflict of interest.

ACKNOWLEDGMENT We thank Dr. P. Thomas for discussion and helpful comments.

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Inhibitory neurosteroids and the GABAA receptor.

γ-Aminobutyric acid type A receptors (GABAARs) are vital proteins that are engaged in regulating neural circuit activity in the central nervous system...
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