Allosteric Modulation of GABAA Receptors via Multiple Drug-Binding Sites Werner Sieghart1 Department of Molecular Neurosciences, Center for Brain Research, Medical University Vienna, Vienna, Austria 1 Corresponding author: e-mail address: [email protected]

Contents 1. 2. 3. 4.

5. 6.


8. 9.

Introduction Structure of GABAA Receptors GABA-Binding Sites Benzodiazepine-Binding Sites 4.1 Interaction of benzodiazepine-binding site ligands with the α+γ  interface of GABAA receptors 4.2 Interaction of benzodiazepine-binding site ligands with additional binding sites at GABAA receptors Picrotoxinin-Binding Sites Binding Sites for Anesthetics 6.1 Binding sites of anesthetics in the transmembrane domain within α or β subunits 6.2 A propofol-binding site between TM1 and TM2 of a single β subunit 6.3 Binding sites for etomidate, barbiturates, and propofol in the transmembrane domain at interfaces between subunits 6.4 A possible propofol-binding site in the intracellular loop 6.5 Steroid-binding sites in the transmembrane α+β interface and in the α1 intrasubunit pocket 6.6 A possible loreclezole-binding site near β2Asn265 at the extracellular end of TM2 6.7 A possible n-octanol-binding site near β2Asn265 6.8 Conclusions on the localization of anesthetic binding sites in GABAA receptors Alcohol-Binding Sites 7.1 Alcohol-binding sites in the transmembrane domain 7.2 Alcohol-binding sites in the extracellular α+β interface of α4/6β3δ receptors Cannabinoid-Binding Site Avermectin B1a-Binding Site

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10. Binding Sites of Ions 11. Conclusion Conflict of Interest References

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Abstract GABAA receptors are ligand-gated ion channels composed of five subunits that can be opened by GABA and be modulated by multiple pharmacologically and clinically important drugs. Over the time, hundreds of compounds from different structural classes have been demonstrated to modulate, directly activate, or inhibit GABAA receptors, and most of these compounds interact with more than one binding site at these receptors. Crystal structures of proteins and receptors homologous to GABAA receptors as well as homology modeling studies have provided insights into the possible location of ligand interaction sites. Some of these sites have been identified by mutagenesis, photolabeling, and docking studies. For most of these ligands, however, binding sites are not known. Due to the high flexibility of GABAA receptors and the existence of multiple drug-binding sites, the unequivocal identification of interaction sites for individual drugs is extremely difficult. The existence of multiple GABAA receptor subtypes with distinct subunit composition, the contribution of distinct subunit sequences to binding sites of different receptor subtypes, as well as the observation that even subunits not directly contributing to a binding site are able to influence affinity and efficacy of drugs, contribute to a unique pharmacology of each GABAA receptor subtype. Thus, each receptor subtype has to be investigated to identify a possible subtype selectivity of a compound. Although multiple binding sites make GABAA receptor pharmacology even more complicated, the exploitation of ligand interaction with novel-binding sites also offers additional possibilities for a subtype-selective modulation of GABAA receptors.

NONSTANDARD ABBREVIATIONS Alphaxalone 3α-hydroxy-5α-pregnane-11,20-dione CGS 9895 2-p-methoxyphenylpyrazolo[4,3-c]quinolin-3(5H)-one DMCM methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate EBOB 1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]octane ELIC ligand-gated ion channel from Erwinia chrysanthemi Fa131 trans-(2S,3R)-3-acetoxy-40 -methoxyflavan Fa173 cis-(2S,3S)-3-acetoxy-30 ,40 -dimethoxyflavan Flumazenil Ro15-1788, ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a] [1,4]benzodiazepine-3-carboxylate GLIC ligand-gated ion channel from Gloeobacter violaceus GluCl glutamate-gated chloride channel from Caenorhabditis elegans Ro15-4513 ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo-1,4benzodiazepine-3-carboxylate TBPS t-butylbicyclophosphorothionate THDOC (3α,5β)-3,21-dihydroxypregnan-20-one

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1. INTRODUCTION GABAA receptors are the major inhibitory transmitter receptors in the brain. They are chloride ion channels that can be opened by GABA and are composed of five subunits. The existence of six α, three β, three γ, one δ, one ε, one θ, one π, and three ρ subunits and their distinct regional and cellular distribution in the brain gives rise to a multiplicity of GABAA receptor subtypes with different subunit composition and distinct pharmacological properties (Olsen & Sieghart, 2008). The majority of GABAA receptors, however, are composed of two α, two β, and one γ2 subunit. GABAA receptors are the site of action of a variety of pharmacologically and clinically important drugs such as benzodiazepines, barbiturates, neuroactive steroids, inhalation and intravenous anesthetics, and convulsants, which allosterically modulate GABA-induced currents via distinct binding sites (Sieghart, 1995). The presence of multiple allosteric binding sites at single GABAA receptors results in an extremely complex pharmacology of these receptors and raises the question where all these binding sites are located. Knowledge on the location and structure of these binding sites is essential for understanding GABAA receptor modulation by these clinically important drugs. In addition, this knowledge as well as that on homologous sites in other GABAA receptor subtypes is a prerequisite for a future structure-based drug design that could dramatically accelerate the development of novel subtypeselective drugs of potential therapeutic use (Rudolph & Knoflach, 2011; Rudolph & Mohler, 2014). In the last couple of years, a variety of biochemical and molecular pharmacological techniques have been applied to identify and locate the various binding sites at GABAA receptors. These techniques include site-directed mutagenesis with functional or ligand-binding analysis, cysteine substitution and modifier accessibility with and without ligand protection, photoaffinity labeling, X-ray crystallography, and homology modeling. In this chapter, our current knowledge on the location and possible structure of various GABAA receptor-binding sites is summarized and the advantages and disadvantages of the various localization techniques are discussed. Although most of our knowledge has been gained by investigating α1β2/3γ2 receptors, the most abundant receptors in the brain, similar binding sites can also be found in most, if not all, GABAA receptors. Differences in homologous binding sites possibly can be used for a selective modulation of specific receptor subtypes.


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2. STRUCTURE OF GABAA RECEPTORS GABAA receptors are composed of five subunits that form the central chloride channel. Each subunit contains a large N-terminal extracellular domain, four transmembrane domains (TMs) each forming an α-helix, a large intracellular loop between TM3 and TM4, and a short extracellular C-terminus (Schofield et al., 1987). Experiments investigating the subunit arrangement of GABAA receptors composed of 2α, 2β, and one γ subunit have indicated that α and β subunits alternate with each other and are connected by a γ subunit (Tretter, Ehya, Fuchs, & Sieghart, 1997). This concept for the first time could be visualized by homology models of the extracellular domain of GABAA receptors using the crystal structure of the acetylcholinebinding protein as a template (Brejc et al., 2001; Cromer, Morton, & Parker, 2002; Ernst, Brauchart, Boresch, & Sieghart, 2003). These models as well as experiments with concatenated subunits (Baumann, Baur, & Sigel, 2002) provided the absolute arrangement of subunits in GABAA receptors (Fig. 1A). Each subunit has a plus (+) and a minus () side. The two GABA-binding sites of the receptors are located at the extracellular β+α  interfaces (Smith & Olsen, 1995), whereas the benzodiazepinebinding site is located at the extracellular α+γ  interface (Ernst et al., 2003; Sigel & Buhr, 1997). Ligand-binding sites at the extracellular interfaces are formed by six “loops.” Loops A, B, and C, are located at the plus (principle) side of each subunit and “loops” D, E, and F, are located at the minus (complementary) side (Ernst et al., 2003). Structural models of the extracellular and transmembrane domains of GABAA receptors then indicated that there are multiple solvent accessible spaces within the structure of GABAA receptors (Ernst et al., 2005). In addition to the five subunit interfaces in the extracellular domain, similar five interfaces are also present in the transmembrane domain between the TM1 of one subunit and the TM3 of another subunit (intersubunit site, Fig. 1B and C). A third type of solvent accessible spaces is present within the four helix bundle of the transmembrane domain of each subunit (intrasubunit site). A fourth type of solvent accessible spaces is present within the ion channel formed by the TM2 of the five subunits (Fig. 1B). All these solvent accessible spaces (pockets) differ from each other in their size and their hydrophilic and hydrophobic properties depending on the types of amino acid residues contributing to their formation (Ernst et al., 2005). In addition, neighboring subunits can influence the conformation of the

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Figure 1 Model structure of the GABAA receptor. (A) Model of the extracellular domain of the GABAA receptor, giving the absolute arrangement for α1, β2, and γ2 containing receptors, the view is from extracellular. The + (plus) and  (minus) sides of the subunits are identified on the inner circumference of the channel. The location of the two GABAbinding sites at the β2+α1 interfaces and the benzodiazepine (BZ)-binding site at the α1+γ2  interface is indicated by arrows. The figure is a modification of the figure in Ernst et al. (2003). (B) Solvent accessible spaces contained in GABAA receptor transmembrane models. The view is from outside the cell with the extracellular domain invisible. The four transmembrane helical domains of each subunit are shown. Solvent accessible spaces are within each transmembrane four helical domain (pale gray) as well as between helix 1 of one subunit and helix 3 of the neighboring subunit (dark gray), as well as within the central ion channel (intermediate gray). (C) Dimer of the extracellular and transmembrane domain of β and α subunits (GABA-binding site) viewed from the outside of the pore. Solvent accessible spaces are within the extracellular interface (intermediate gray), within the continuation of this space in the transmembrane domain between two 4 helix bundles (dark gray), within the four helix bundle of each subunit (pale gray). Panels (B) and (C) are modifications of figures shown in Ernst, Bruckner, Boresch, and Sieghart (2005).

pockets via subunit–subunit interactions (Sections 3 and 7.2), indicating that even pockets formed by the same four transmembrane helices of for instance the two α or two β subunits of GABAA receptors could be different from each other. Since some of them might accommodate more than one drug at distinct positions, these solvent accessible spaces add up to more than 16 distinct binding sites. The solvent accessible spaces probably render the receptor extremely flexible and are necessary for allowing


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conformational changes during the GABA-activated opening of the channel. Drugs fitting into or binding to any one of these multiple allosteric binding sites can stabilize or induce certain conformational changes of the receptor, thus enhancing or reducing GABA-induced chloride flux. By that, GABA and any allosteric modulator binding to the receptor might change the size and shape of other pockets differentially. This state-dependent conformational pocket change could result in state-dependent drug binding (see below) and, thus, again might increase the number of distinct binding sites within the receptor. Furthermore, ligand-bound crystal structures of the bacterial homologues of GABAA receptors isolated from Gloeobacter violaceus (GLIC; Bocquet et al., 2009; Hilf & Dutzler, 2009) and Erwinia chrysanthemi (ELIC; Hilf & Dutzler, 2008), as well as structures of a glutamate-gated channel from the nematode Caenorhabditis elegans (GluCl; Hibbs & Gouaux, 2011), and the recently published crystal structure of the homooligomeric β3 GABAA receptor (Miller & Aricescu, 2014), indicated the existence of additional ligand-binding sites in the transmembrane and extracellular domains (for location, see Howard, Trudell, & Harris, 2014; Spurny et al., 2012), as do mutagenesis studies and more specific modeling studies (Baur et al., 2013). Finally, we currently have only very limited information (Unwin, 2005) on the structure of the intracellular part of the receptors formed by the large intracellular loops between TM3 and TM4 of each subunit. It can be assumed that there also will be possible drug-binding sites. Drugs interacting with such sites might either elicit a distinct conformation of the receptor, thus leading to a modulation of GABA-induced currents (Moraga-Cid, Yevenes, Schmalzing, Peoples, & Aguayo, 2011) or interfere with the binding of associated proteins that regulate GABAA receptor function and location (Chen & Olsen, 2007). So our current knowledge on the structure of GABAA receptors indicates the existence of many more potential drug-binding sites than previously expected. Some of these sites have clearly defined ligands, and the location of these sites within the receptor, and in a few cases their tentative interaction with some ligands, has been more or less unequivocally identified. These sites will be shortly discussed below. For other potential-binding sites, however, no ligands are known. In addition, the sites of action of most of the literally hundreds of compounds from different structural classes that allosterically modulate GABAA receptors are not yet identified.

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3. GABA-BINDING SITES Over the years, considerable insights into the GABAA receptor orthosteric binding pocket have been obtained from site-directed mutagenesis studies, from studies using [3H]muscimol for photolabeling, or from receptor-binding studies using a range of GABA-site ligands (Petersen et al., 2013; Smith & Olsen, 1995). It is now clear that the GABA-binding site is located at the extracellular β+α  interface of GABAA receptors (Fig. 1A). Since the majority of GABAA receptors contain 2α, 2β, and one γ subunit, these receptors also contain two β+α  interfaces and thus two GABA-binding sites. Occupancy of both sides greatly enhances the probability of opening the intrinsic channel (Baumann, Baur, & Sigel, 2003). However, due to the low sequence identity of GABAA receptors and its first template, the crystal structure of the acetylcholine-binding protein, the first model structures obtained (Cromer et al., 2002; Ernst et al., 2003) did not provide sufficient information on the exact way of binding of GABA into the orthosteric binding sites. Additional crystal structures of GLIC, ELIC, and GluCl then allowed to obtain more reliable models of the GABA site of GABAA receptors using combinations of the available templates (Bergmann, Kongsbak, Sorensen, Sander, & Balle, 2013; Sander et al., 2011). In addition, recently a GABA-bound crystal structure of ELIC was published, providing some information on the direct interaction of GABA with amino acid side chains of the receptor (Spurny et al., 2012). Due to differences in the amino acid sequences between GluCl, ELIC, and GABAA receptors, however, as well as due to the fact that multiple amino acid residues in the GABA pocket possibly could interact with GABA, the binding modes of GABA so far obtained not necessarily are identical with those observed in GABAA receptors. Crystal structures of GABA-bound GABAA receptors are therefore required for a final confirmation of the interaction of GABA with its binding site in native receptors. Interestingly, concatenated and point-mutated GABAA receptors indicated that the two GABA-binding sites of α1β2γ2 receptors exhibit slightly different properties for agonists but similar properties for competitive antagonists (Baumann et al., 2003). These data thus support the conclusion that the different flanking subunits (γ and β, α and γ) of these otherwise identical binding sites at the two β+α  interfaces (Fig. 1A) cause a slightly different conformation or signal transduction of these sites. Further experiments will


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have to clarify whether these differences also result in a distinct pharmacology of these two binding sites. In addition to the two GABA-binding sites each one located at one of the two β+α  interfaces of the most abundant GABAA receptors composed of 2α, 2β, and one γ subunit, other so far only weakly characterized binding sites might exist at some receptors composed of not as abundant subunit combinations. Thus, studies with concatenated α1β3δ GABAA receptors have indicated that there might be several possibilities for the incorporation of a δ subunit. In one of these receptors, the δ subunit might contribute to a novel GABA-binding site (Kaur, Baur, & Sigel, 2009). This conclusion is supported by the finding that α6βδ (Hadley & Amin, 2007) and α4β1/3δ (Karim et al., 2012) receptors exhibit nanomolar- and micromolar-affinity GABA-binding sites and that the latter receptor might form a novel GABA-binding site at the δ subunit interface (Karim et al., 2012).

4. BENZODIAZEPINE-BINDING SITES 4.1. Interaction of benzodiazepine-binding site ligands with the α+γ 2 interface of GABAA receptors Benzodiazepines, such as chlordiazepoxide or diazepam, were introduced into clinical use in the 1960s and due to their anxiolytic, anticonvulsant, sedative hypnotic, and muscle relaxant properties soon became the most commonly prescribed drugs in therapeutic use. After the first cloning of GABAA receptor subunit cDNAs (Schofield et al., 1987), [3H]flunitrazepam-binding studies as well as studies investigating the electrophysiological effects of benzodiazepines at recombinant GABAA receptor subtypes containing different subunit combinations indicated that benzodiazepines require a combination of α, β, and γ subunits for their interaction with GABAA receptors (Pritchett et al., 1989). Studies using the benzodiazepines [3H]flunitrazepam or [3H] Ro15-4513 as photoaffinity labels, as well as site-directed mutagenesis studies, identified amino acid residues in α and γ subunits that seem to be important for benzodiazepine action (Sigel & Buhr, 1997). These residues could be put into place in a three-dimensional structure when the first homology models of the extracellular domain of GABAA receptors were generated (Ernst et al., 2003). Using multiple templates from various crystal structures, a binding mode of diazepam and its structural analogues could be identified that is consistent with most of the experimental results available and that also could be used for structure and fragment-based drug discovery (Richter et al., 2012). In the meantime, hundreds of ligands from >41 structural

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classes have been identified that are able to bind to the benzodiazepinebinding site of GABAA receptors and only a few of these distinct structural classes can also bind to the same pocket conformation as diazepam (Richter et al., 2012). For a final confirmation of the validity of these model structures, therefore, again crystal structures of various ligand-bound GABAA receptors are required that also will provide information on the conformational space of the benzodiazepine-binding site of GABAA receptors.

4.2. Interaction of benzodiazepine-binding site ligands with additional binding sites at GABAA receptors 4.2.1 Benzodiazepine-binding sites possibly located in the transmembrane domain Some well-known benzodiazepine site ligands, however, bind not only to the classical benzodiazepine site at the extracellular α+γ  interface but also to other binding sites in GABAA receptors. Thus, diazepam (Walters, Hadley, Morris, & Amin, 2000) is able to modulate α1β2γ2 GABAA receptors with a nanomolar and a micromolar component. The nanomolar component depends on the presence of the γ2 subunit and is inhibited by the benzodiazepine site antagonist flumazenil (Ro15-1788, ethyl-8-fluoro5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3carboxylate). The micromolar component of diazepam action enhances GABA-induced currents of both α1β2 and α1β2γ2 receptors and this effect is not blocked by flumazenil (Walters et al., 2000). Mutation at residues within the second transmembrane domains of α, β, and γ subunits, proven important for the action of anesthetics, abolishes the micromolar, but not the nanomolar component of diazepam action. These data support at least two mechanisms of action of diazepam. The classical modulation via the highaffinity benzodiazepine-binding site and a second low potency modulation possibly elicited via an anesthetic binding site or a site that mediates its action via amino acid residues important for anesthetic action. This second mechanism of action might also explain the anesthetic properties of diazepam that in addition to its application as an anxiolytic, sedative hypnotic, muscle relaxant, and anticonvulsant drug is routinely used as premedication for surgical procedures (Walters et al., 2000). Some β-carbolines also exhibit a biphasic action at nanomolar and micromolar concentrations (Stevenson, Wingrove, Whiting, & Wafford, 1995; Thomet, Baur, Scholze, Sieghart, & Sigel, 1999). Whereas the high-affinity binding of β-carbolines, such as DMCM (methyl-6,7-dimethoxy-4-ethyl-βcarboline-3-carboxylate), occurs at the benzodiazepine-binding site, the


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β2/3 subunit dependence of the low-affinity effect of these compounds as well as site-directed mutagenesis experiments suggested that DMCM and other β-carbolines exhibit a second interaction with a binding site previously identified for the sedative and anticonvulsant loreclezole (Wingrove, Wafford, Bain, & Whiting, 1994) and the anesthetic etomidate (Belelli, Lambert, Peters, Wafford, & Whiting, 1997). In contrast to diazepam, that exhibits a positive allosteric modulation via the high affinity as well as via the low-affinity site, DMCM exhibits a negative allosteric modulation via the high-affinity site and a positive allosteric modulation via the low-affinity site (Stevenson et al., 1995). The opposite actions of this compound via the different binding sites might explain why DMCM, in contrast to diazepam, does not produce anesthesia at high concentrations. 4.2.2 Benzodiazepine-binding sites at the α+β 2 interface Diazepam and other classical benzodiazepines, however, seem to have a third site of action at GABAA receptors that negatively modulates the classical benzodiazepine-binding site (Baur et al., 2008; Walters et al., 2000). Results with the benzodiazepine flurazepam indicated that this negatively interacting site may be located at the α1+β2  subunit interface pseudosymmetrically to the site for the classical benzodiazepines located at the α1+γ2  interface (Baur et al., 2008). The extracellular α1+β3  interface of GABAA receptors recently was investigated in more detail as a potential drug-binding site (Ramerstorfer et al., 2011). It was demonstrated that the anxiolytic pyrazoloquinolinone CGS 9895 (2-p-methoxyphenylpyrazolo[4,3-c]quinolin-3(5H)-one), which has previously been demonstrated to exhibit a high affinity for the benzodiazepine-binding site of GABAA receptors, was able to strongly enhance GABA-induced currents also at α1β3 receptors that do not exhibit a classical benzodiazepine-binding site. A steric hindrance procedure indicated that the binding site of this compound is located at the α1+β3  interface at a position homologous to that of the classical benzodiazepinebinding site. Other experiments demonstrated that CGS 9895 acts as a high-affinity null modulator (antagonist) at the benzodiazepine-binding site and as a low potency positive allosteric modulator at the α1+β3  interface (Ramerstorfer et al., 2011). Thus, most of the actions of CGS 9895 and other pyrazoloquinolinones are mediated via its low-affinity binding site at the α1+β3  interface, and not via the high-affinity benzodiazepine-binding site as previously assumed. As expected from its location, the efficacy of various pyrazoloquinolinones strongly depends on the type of α and β subunits

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present within the receptor (Varagic, Ramerstorfer, et al., 2013), and this property makes it an excellent target for the development of novel receptor subtype-selective drugs (Sieghart, Ramerstorfer, Sarto-Jackson, Varagic, & Ernst, 2012). To investigate a possible identity of the CGS 9895 binding site and the negatively modulating flurazepam-binding site discussed above (Baur et al., 2008), that both should be located at the extracellular α+β  interface, the effects of flurazepam were investigated at α1β3 receptors (Ramerstorfer et al., 2011). In the absence of a classical benzodiazepine-binding site at these receptors, this compound exhibited no effects up to 1 μM concentrations and dose-dependently inhibited GABA-induced currents at concentrations between 10 μM and 1 mM. The inhibitory effect of flurazepam, however, could not be blocked by steric hindrance experiments that were able to block the action of CGS 9895, indicating that this inhibitory effect of flurazepam is not mediated via the extracellular α1+β3  interface. Flurazepam interaction with the extracellular α1+β2 site (Baur et al., 2008) but not with the extracellular α1+β3  site (Ramerstorfer et al., 2011) thus possibly indicates a β subunit-specific interaction of this ligand at this interface. Recently, it was demonstrated that the imidazobenzodiazepine Ro15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo-1, 4-benzodiazepine-3-carboxylate) not only is a high-affinity ligand at the benzodiazepine-binding site but also specifically and with high affinity interacts with sites located at the α6+β3  or α4+β3  interfaces, where it inhibits some of the actions of ethanol (Wallner, Hanchar, & Olsen, 2014). This interaction will be discussed more extensively in Section 7.2. 4.2.3 Benzodiazepine binding to the GABA-binding site (β+α 2 interface) Inhibition by flurazepam of GABA-induced currents in α1β3 receptors, however, might have been mediated by a direct interaction of flurazepam with the GABA-binding site. This conclusion was supported by radioligand-binding studies indicating that the binding of 10 nM [3H] muscimol to rat cerebellar membranes can be completely inhibited by high concentrations (300 μM to 30 mM) of flurazepam in a concentrationdependent way (W. Sieghart and J. Ramerstorfer, unpublished results). A possible direct interaction of flurazepam with the GABA-binding site of GABAA receptors at high concentrations is also supported by ligandbound crystal structures of ELIC (Spurny et al., 2012). As with the


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hetero-oligomeric GABAA receptors, flurazepam at 50 μM concentrations potentiated currents induced by 10 mM GABA in the homo-pentameric ELIC up to 180%, but inhibited GABA-induced currents at higher concentrations. Crystal structures of ELIC formed in the presence of 10 mM GABA and 50 μM flurazepam identified a GABA-binding site at subunit interfaces at a position equivalent to the GABA-binding site in GABAA receptors (see Section 3; Spurny et al., 2012). Interestingly, under these conditions, flurazepam simultaneously was bound to a novel intrasubunit-binding site in the extracellular domain (see Section 4.2.4). Using 10 mM GABA and much higher (10 mM) concentrations of a bromo-analogue of flurazepam (Br-flurazepam), or 10 mM GABA and 10 mM of the benzodiazepine site ligand zopiclone during crystallization of ELIC, two other crystal structures of ELIC could be obtained that both indicated benzodiazepine-binding sites at subunit interfaces that partially overlap with the recognition site for GABA in the crystal structure at the low-flurazepam concentration (Spurny et al., 2012). Presumably due to the higher affinity of Br-flurazepam and zopiclone (micromolar) than GABA (millimolar) for a partially overlapping binding site, no GABA could be found in these crystal structures. Although the binding site of Br-flurazepam in ELIC at high concentrations in principle resembled that at the α+γ  interface of GABAA receptors, its binding pose within this pocket was different from that of benzodiazepines in the benzodiazepine-binding site. Presumably, the divergent amino acid sequence of ELIC and that of the high-affinity benzodiazepine site at the α+γ  interface of GABAA receptors caused differences in the pocket architecture and resulted in a benzodiazepine-binding pose in ELIC that is different from that in GABAA receptors. This not necessarily is the case for all ligands binding to the ELIC interface. Interestingly, although a racemic mixture of zopiclone was used for cocrystallization with ELIC, a preferential fit in electron density of the S-enantiomer of zopiclone was observed (Spurny et al., 2012). This enantioselectivity corresponds to what is known from S-zopiclone binding to the high-affinity benzodiazepine-binding site in GABAA receptors (Hanson, Morlock, Satyshur, & Czajkowski, 2008). Detailed structural analysis indicated that zopiclone in this crystal structure is involved in key interactions with conserved aromatic residues that are also involved in the binding of benzodiazepines in the benzodiazepine pocket of GABAA receptors. This suggests that zopiclone might adopt a binding pose in ELIC that possibly mimics the ligand orientation at the α+γ  interface of GABAA receptors. All these data indicate that at least some benzodiazepine site

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ligands can interact not only with the benzodiazepine site at the α+γ  interface, but at high concentrations also with the GABA-binding site at the β+α  interface or the pyrazoloquinolinone site at the α+β  interface, supporting the structural similarity of the extracellular pockets of GABAA and other cys-loop receptors. Depending on the structure of the pocket and on that of the ligand, the ligand might be able to bind to the GABA site or to sites located at other interfaces in a way similar to or different from that observed at the benzodiazepine site. 4.2.4 Benzodiazepine binding to an extracellular intrasubunit site Whereas high concentrations of flurazepam resulted in a crystal structure in which flurazepam is bound to the extracellular interface of ELIC thus displacing GABA from its binding site, crystallization performed in the presence of low concentrations of flurazepam resulted in crystal structures exhibiting a distinct localization of flurazepam (Spurny et al., 2012). Under these conditions, flurazepam is bound to an intrasubunit cavity facing the channel vestibule of the extracellular domain. This site is localized at the same height as the GABA intersubunit-binding site, but lies opposite the inner walls formed by loop B (+ side) and loop D ( side) of the neighboring GABA-recognition site (Spurny et al., 2012). This site is ideally positioned to modulate GABA function allosterically in ELIC, and site-directed mutagenesis followed by the investigation of the flurazepam modulation of GABA currents in ELIC supported this conclusion. An intrasubunit pocket exactly matching the intrasubunit benzodiazepine site in ELIC has been identified in the crystal structure of the muscle α1 nACh receptor subunit (Dellisanti, Yao, Stroud, Wang, & Chen, 2007; Dey & Chen, 2011) and was used in a structure-based drug-design approach for nACh receptor modulators (Dey & Chen, 2011). The possible existence of a similar intrasubunit site in eukaryote GABAA receptors is supported by a striking sequence similarity of residues lining the intrasubunit-binding site (Spurny et al., 2012). Further experiments will have to investigate whether such sites are used and have a functional role in GABAA receptors. 4.2.5 High-affinity flunitrazepam binding to a “non-benzodiazepine” site at α6β2γ2 receptors An additional receptor subtype-selective interaction with a so far unidentified binding site was observed with the benzodiazepine flunitrazepam. This compound is a positive allosteric modulator acting via classical benzodiazepine-binding sites at α1βγ2, α2βγ2, α3βγ2, and α5βγ2 receptors,


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but exhibits a very low potency for displacing [3H]Ro15-4513 binding at α6β2γ2 receptors (Sieghart, 1995). Several years ago, it was demonstrated that flunitrazepam nevertheless is able to exhibit a high-affinity binding to α6β2γ2 receptors (Kd ¼ 8.7 nM, as compared to 3.3 nM for α1β2γ2 receptors; Hauser, Wetzel, Berning, Gerner, & Rupprecht, 1997). This highaffinity binding of [3H]flunitrazepam to α6β2γ2 receptors was only weakly inhibited by the benzodiazepine site antagonist flumazenil or other benzodiazepine site ligands and did not compete with the high-affinity Ro154513 binding site (presumably the classical benzodiazepine-binding site) at α6β2γ2 receptors, or the high-affinity Ro15-4513/ethanol-binding site at α4/6β3δ receptors (Hanchar et al., 2006). Flunitrazepam elicited a negative allosteric modulatory (inverse agonist) effect at α6β2γ2 receptors and this effect was dependent on the concentration of GABA. The location of the high-affinity [3H]flunitrazepam-binding site at α6β2γ2 receptors, which obviously is different from the classical benzodiazepine-binding site, was not investigated in this study.

5. PICROTOXININ-BINDING SITES After the identification of GABAA receptors as being activated by GABA and inhibited by bicuculline (Curtis, Phillis, & Watkins, 1959), picrotoxin was identified as an additional noncompetitive inhibitor of these receptors (Curtis, Duggan, & Johnston, 1969; Johnston, 1978). This inhibitor was not as selective for GABAA receptors as bicuculline and at higher concentrations also inhibited glycine receptors as well as other receptors from the same superfamily. The identification of high-affinity radioligands for the picrotoxin-binding site ([3H]dihydropicrotoxin, [35S]tbutylbicyclophosphorothionate (TBPS), [3H]1-(4-ethynylphenyl)-4-npropyl-2,6,7-trioxabicyclo[2.2.2]octane (EBOB), and others; Chen, Durkin, & Casida, 2006; Olsen, Ticku, & Miller, 1978) led to a multiplicity of studies investigating the pharmacology of this binding site. Identification of a point mutation in a GABA-gated chloride channel of Drosophila melanogaster (RDL; the product of the “resistance to dieldrine locus”) then provided the first evidence for a possible location of the picrotoxinin site within the channel lumen (Hosie, Baylis, Buckingham, & Sattelle, 1995). Results indicated that the channel-blocking activities of the insecticides dieldrine and fipronil, as well as that of picrotoxinin, were reduced by this mutation. Other results indicated that this mutation also dramatically reduced the binding of [3H]EBOB (Cole, Roush, & Casida, 1995). Together, these studies

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indicated that picrotoxinin, the biologically active component of picrotoxin, as well as several major insecticides and convulsants bind within the channel lumen of GABAA, glycine, and RDL receptors. Use of cysteine mutagenesis combined with covalent chemical modification and electrophysiological studies in the absence or presence of picrotoxinin then allowed identifying amino acid residues within the channel as possible picrotoxininbinding sites (Perret et al., 1999; Xu & Akabas, 1996). Picrotoxinin and TBPS interact with both resting and GABA-bound receptors, but their affinity for the latter is about 10 times greater than that for the former. This seems to be largely due to a markedly increased association rate to the receptor (Dillon, Im, Carter, & McKinley, 1995), probably caused by GABA-induced channel opening that enhances ligand access to the channel lumen. Once bound, however, these convulsants stabilize an agonist-bound shut state (Newland & Cull-Candy, 1992). Binding of these convulsants occurs between residues A20 , T60 , and L90 of the human homopentameric β3 receptor (Chen et al., 2006) according to a widely used nomenclature that numbers the amino acid residues of the five TM2s forming the channel and that starts with position 20 that occupies the N-terminal cytoplasmic amino acid residue and ends with the C-terminal extracellular end of the pore (Fig. 1C). Modeling studies indicated that all these convulsants fit to the 20 to 90 pore region, forming hydrogen bonds with the T60 hydroxyl groups and hydrophobic interactions with A20 , T60 , and L90 alkyl substituents, thereby blocking the channel (Chen et al., 2006). Similar results were also obtained for hetero-oligomeric rat GABAA receptors composed of α1β2γ2 subunits, and interaction of picrotoxinin with three adjacent TM2 60 residues via hydrogen bonds seems to be sufficient for inhibition (Erkkila, Sedelnikova, & Weiss, 2008). In addition, it was demonstrated that any one of the five subunits carrying a TM2 60 mutation can impart picrotoxin resistance (Sedelnikova, Erkkila, Harris, Zakharkin, & Weiss, 2006). The recently published crystal structure of GluCl (Hibbs & Gouaux, 2011) confirms the binding of picrotoxinin within the channel. Electron density in picrotoxin-soaked GluCl crystals was apparent at a position near the cytosolic side of the transmembrane pore (Fig. 1C). Picrotoxin directly occluded the pore near its cytosolic base at the 20 Thr and the 20 Pro side chains. The more cytosolic position of picrotoxin in GluCl might have been caused by differences in the structure of GluCl and GABAA receptors or might reflect a different state of GluCl (open state) and GABAA receptors (desensitized state? see below) under the conditions of measurements.


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Other studies are consistent with an additional binding site of picrotoxin and TBPS at residues 150 and 170 of the TM2 of GABAA receptor α1 subunits, which are located at the extracellular end of the channel (Perret et al., 1999). A second binding site at a similar location for picrotoxinin has also been demonstrated for glycine receptors (Dibas, Gonzales, Das, BellHorner, & Dillon, 2002) or for homo-oligomeric ρ receptors (Carland, Johnston, & Chebib, 2008). While these results support a direct occlusion of the channel, kinetic measurements and their interpretation argue for an allosteric mechanism of action of picrotoxinin (stabilization of a desensitized state; for discussion, see Korshoej, Holm, Jensen, & Lambert, 2010). Whether both concepts can be combined have to be clarified by further experiments: the channel must be open for picrotoxin to reach its binding site. Whether the receptor has to be desensitized in order for picrotoxin to bind, or its binding actually induces desensitization, is not presently resolved (Korshoej et al., 2010). In addition, the possible involvement of the additional binding site of picrotoxinin at residues 150 and 170 of the TM2 of GABAA receptor α1 subunits (Perret et al., 1999), might also be interesting to investigate. Picrotoxinin has to pass this site to reach the cytosolic end of the channel where the binding site for its blocking action seems to be located. Although many compounds have been identified that are able to inhibit picrotoxinin or TBPS binding, not all of these compounds also interact with the picrotoxinin-binding site. It has to be stressed that the pharmacology of the picrotoxinin/TBPS-binding site of GABAA receptors is extremely complex due to the location of the site within the channel. Higher concentrations of GABA causing an increased chloride flux, or any compound that opens the channel or enhances GABA-induced channel opening increase the dissociation of these ligands from their binding site, thus causing an apparent displacement. A distinction between a competitive and allosteric displacement can only be made by measuring the drug-induced dissociation rate of the radioactive ligand (Maksay & Ticku, 1985). In contrast to a competitive displacement that does not change the dissociation rate of the radioactive ligand, allosteric displacement induced by channel opening increases the dissociation rate.

6. BINDING SITES FOR ANESTHETICS Drugs acting via the benzodiazepine site at the extracellular α+γ  interface, or the pyrazoloquinolinones acting via the extracellular

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α+β  interface, can only modulate GABAA receptors but not directly open the channel in the absence of GABA. In contrast, barbiturates, neuroactive steroids, and anesthetics at low concentrations can enhance GABA-induced currents, whereas at higher concentrations, they are able to directly activate GABAA receptors in the absence of GABA. In addition, receptor-binding studies indicated biphasic or even multiphasic dose–response curves for these compounds, suggesting their interaction with more than one binding site at GABAA receptors (Sieghart, 1995). In this chapter, the binding sites for the inhalation anesthetics halothane, enflurane, isoflurane, and chloroform, for the intravenous anesthetics, pentobarbital, etomidate, and propofol, the steroids THDOC ((3α,5β)3,21-dihydroxypregnan-20-one) and alphaxalone (3α-hydroxy-5αpregnane-11,20-dione), as well as those for the anticonvulsant loreclezole are discussed. Although not all of these anesthetics bind to the same binding site, their main sites of action seem to be within the transmembrane domain of GABAA receptors and to be located close to each other. However, binding sites in the extracellular and intracellular domain also have been suggested. Again, several methods have been used for the identification of anesthetic binding sites. In contrast to the GABA, benzodiazepine, or picrotoxinin-binding sites discussed above, for which different methods resulted in a consensus localization of the respective binding sites, here different methods led to partially different conclusions. Thus, a consensus on the location of the binding sites for all these anesthetics has not yet been reached. In this chapter, the results obtained with different methods will be compared, possible explanations for discrepant results will be given, and the chapter will be concluded with a discussion of the possible sites of action of these drugs.

6.1. Binding sites of anesthetics in the transmembrane domain within α or β subunits Studies investigating the effects of site-directed mutagenesis on drug modulation of GABAA receptors have identified several amino acid residues in the α and β subunit transmembrane domains that seem to be important for the action of the investigated drugs. Over time evidence accumulated that some of these residues might directly contribute to binding pockets for anesthetics. Results indicated that inhalation anesthetics such as isoflurane, enflurane, halothane, chloroform, and ethanol might elicit their action by binding to the pocket within the four α-helices of the α subunit


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transmembrane domain ( Jenkins et al., 2001; Mihic et al., 1997; Yamakura, Bertaccini, Trudell, & Harris, 2001), whereas intravenous anesthetics such as propofol and etomidate might interact with the homologous pocket of β subunits (Bali & Akabas, 2004; Belelli et al., 1997; Krasowski, Nishikawa, Nikolaeva, Lin, & Harrison, 2001; Richardson et al., 2007). Since most of the GABAA receptors are composed of 2α, 2β, and one γ subunit, this binding within α or β subunits suggests the existence of at least two binding sites for each of these anesthetics that not necessarily exhibit the same affinity for these drugs (see below). The location of anesthetic binding sites in intrasubunit pockets was supported by two recent crystal structures of proteins homologous to GABAA receptors. Thus, a crystal structure of GLIC cocrystallized with anesthetics indicated that propofol and desflurane are binding to the intrasubunit transmembrane pockets of this receptor (Nury et al., 2011). Whereas propofol binds at the entrance of the cavity, the smaller, more flexible, desflurane binds deeper inside. In another study (Spurny et al., 2013), a crystal structure of ELIC in complex with bromoform revealed anesthetic binding sites in the channel pore and in novel sites in the transmembrane and extracellular domain, suggesting that general anesthetics allosterically modulate channel function via multisite binding. It has to be stressed, however, that in contrast to GABAA receptors, which are positively modulated by propofol and other anesthetics, most nACh receptors as well as GLIC or ELIC are inhibited by these compounds. Although it is possible that binding sites mediating inhibition of currents in one receptor might elicit enhancement of currents in a homologous receptor, this not necessarily is true. In addition, recently it was demonstrated that mutation at the F140 site in GLIC TM2 that moves ligands between intraand intersubunit sites turns desflurane and chloroform from inhibitors to potentiators (Bromstrup, Howard, Trudell, Harris, & Lindahl, 2013). This was explained by the existence of potentiating and inhibiting sites in ligandgated ion channels. The overall modulation of receptors possibly might be generated by the net effect of ligand binding to an intersubunit potentiating (see Section 6.3) and an intrasubunit inhibitory site (Bromstrup et al., 2013). Very recently, the crystal structure of the human homo-oligomeric β3 GABAA receptor has been published (Miller & Aricescu, 2014). This was the very first crystal structure of a GABAA receptor and for sure will become important for future drug localization and receptor modeling studies. It demonstrated some similarities and differences to the previously published structures of GLIC, ELIC, and GluCl. Unfortunately, however,

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cocrystallization with anesthetics was not reported in this study, although anesthetics readily bind to (Davies, Kirkness, & Hales, 1997; Slany, Zezula, Tretter, & Sieghart, 1995) and activate this receptor (Cestari, Uchida, Li, Burt, & Yang, 1996). A structure of the β3 receptor cocrystallized with anesthetics is eagerly awaited and might clarify some of the discrepancies on the localization of anesthetics discussed in this chapter.

6.2. A propofol-binding site between TM1 and TM2 of a single β subunit Recently, a new propofol analogue photolabeling reagent (ortho-propofol diazirine, o-PD) was used to identify the propofol-binding site of homooligomeric β3 and hetero-oligomeric α1β3 GABAA receptors (Yip et al., 2013). The propofol photolabel was covalently bound to β3H267. A homology model based on the crystal structure of GluCl indicated that the binding site was located in a cavity within a single β subunit between TM1 and TM2 at the interface between the transmembrane domains and the extracellular domain although there was also an interaction with the main chain at the top of TM2 in the neighboring subunit (Fig. 1B). This cavity is also close to known determinants of anesthetic sensitivity in the transmembrane segments TM1 and TM2, but is clearly different from that of the etomidate-binding site at the β+α  transmembrane interface and is consistent with an allosteric interaction of etomidate and propofol (Yip et al., 2013).

6.3. Binding sites for etomidate, barbiturates, and propofol in the transmembrane domain at interfaces between subunits 6.3.1 Etomidate-binding site at the transmembrane β+α 2 interfaces Using two different photo-incorporable etomidate derivatives, which retain anesthetic potency in vivo and enhance GABAA receptor function in vitro, two amino acid residues, α1Met-236 in the TM1 helix of α1 subunits and β3Met-286 in the TM3 helix of β3 subunits, were irreversibly labeled during allosteric modulation as well as direct opening of the channel by etomidate. These residues are located in the upper part (close to the extracellular domain) of the transmembrane domain of GABAA receptors at the β+α  interface, and thus, below the GABA-binding site (Chiara et al., 2012; Li et al., 2006). Photolabeling of etomidate at this interface seemed to be competitively inhibited not only by etomidate (Chiara et al., 2012)


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but also by isoflurane (Li, Chiara, Cohen, & Olsen, 2010) suggesting that at least this inhalation anesthetic (also) interacts with the same binding site as etomidate at the β+α  interface. Photo-incorporation of etomidate, however, seemed to be allosterically modulated by neurosteroids, barbiturates, and propofol, and not modulated at all by n-octanol or ethanol (for discussion, see Olsen & Li, 2011), indicating that these other drugs do not bind to the transmembrane β+α  interface, or at least bind to a site not overlapping with the etomidate-binding site. A similar etomidate-binding site obviously is also located at the transmembrane β3+β3  interface of α1β3 receptors (Chiara et al., 2012), and this is consistent with the observation that etomidate allosterically modulates homo-oligomeric β3 receptors (Slany et al., 1995). Interestingly, some of these results also indicate that binding of etomidate to the β3 +β3  interface in α1β3 receptors can only occur when the β3+α1  interface is not occupied by etomidate (Chiara et al., 2012). In addition, activation of a receptor by GABA enhances the affinity of etomidate for its binding site (Stewart et al., 2013). Both results support the conclusion that conformational changes elicited by GABA or allosteric modulators can change the structure of at least some of the remaining pockets. 6.3.2 Barbiturate-binding sites at the transmembrane α+β 2, γ+β 2, and β+α 2 interfaces In another study (Chiara et al., 2013), a photoreactive barbiturate was used to identify the barbiturate-binding sites of GABAA receptors. This compound did not photolabel the etomidate sites at the β+α  interface, but instead photolabeled sites at the α+β  and γ+β  interfaces in the TM domain (Chiara et al., 2013). These sites, like the etomidate site, are located at subunit interfaces near the synaptic side of the transmembrane domain. Whereas R-etomidate seems to bind with >100-fold selectivity to the two β+α  interfaces, the photoactive barbiturate binds with >50-fold selectivity to the α+β  and γ+β interfaces in the TM domain, and at the concentration used did not bind to the two β+α  interfaces. The two classes of sites, however, are not simply “etomidate” or “barbiturate” sites. Depending on the structure of the investigated barbiturate- or etomidate-analogues, they exhibit either selectivity for the one or for the other sites, or no selectivity at all (Chiara et al., 2013). 6.3.3 Propofol-binding sites at the transmembrane α+β 2, γ+β 2, and β+α 2 interfaces Propofol was able to inhibit photolabeling of both the photoreactive barbiturate and the photoreactive etomidate, but it seemed to have little

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selectivity for either site (Chiara et al., 2013). These data thus suggest that propofol can bind to at least four binding sites in the transmembrane domain of α1β3γ2 receptors (the α+γ  transmembrane interface was not investigated in this study). Interestingly, the potentiation and direct activation by propofol, which has little or no subunit interface selectivity, is best fit with a model that requires three equivalent binding sites, whereas etomidate only requires two (Ruesch, Neumann, Wulf, & Forman, 2012; Rusch, Zhong, & Forman, 2004). Alphaxalone, however, did not bind to either site, indicating that neurosteroids might bind near these intersubunit anesthetic binding sites but more at the lipid interface (Chiara et al., 2013). These conclusions were confirmed and extended ( Jayakar et al., 2014) by using a photoreactive analogue of propofol (AziPm) different from that used by Yip et al. (2013) and with a much broader reactivity against various amino acid residue types. After applying this compound at heterologously expressed human α1β3 receptors, protein microsequencing identified amino acid residues β3Met-286 and α1Met-236 at the β3+α1  transmembrane interface, and photolabeling of these residues could be inhibited by AziPm, propofol, and etomidate, as well as by the photoreactive barbiturate discussed above. These results provide further evidence that propofol modulation of GABAA receptor function results from propofol binding to the transmembrane intersubunit sites. In this study that already used structural models based on the crystal structure of the transmembrane domain of the homo-oligomeric β3 receptors (Miller & Aricescu, 2014), no evidence was found for [3H]AziPm photolabeling of GABAA receptor amino acids that would be located in intrasubunit-binding pockets of GABAA receptors. Interestingly, however, in affinity purified GLIC, AziPm covalently photolabeled three amino acid residues in proximity to the residues that are in contact with propofol in the intrasubunit pocket of the GLIC crystal structure (Nury et al., 2011) and labeling could be inhibited by propofol (Chiara et al., 2014). These data indicate that the photolabeling results obtained with this compound in GABAA receptors seem to be reliable and that the intrasubunit propofol-binding site in GLIC seems not to be present in GABAA receptors (see discussion in Section 6.1).

6.4. A possible propofol-binding site in the intracellular loop Recently, the large intracellular loop of α1β2 GABAA receptors was screened using the alanine replacement technique, and the effect of these mutations on the sensitivity to propofol was studied (Moraga-Cid et al.,


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2011). Alanine mutation of a conserved phenylalanine residue within the large intracellular loop of the subunits significantly reduced propofol enhancement, whereas the sensitivity to other allosteric modulators such as alcohols, etomidate, trichloroethanol, and isoflurane was not changed by this mutation. This residue influencing the sensitivity to propofol might thus be either a propofol-binding site located near this residue in the intracellular loop or might stabilize the transduction of the propofol effect elicited via a different site (Moraga-Cid et al., 2011).

6.5. Steroid-binding sites in the transmembrane α+β2 interface and in the α1 intrasubunit pocket Site-directed mutagenesis studies identified amino acid residues α1Thr236 and α1Gln241 in the α1 subunit transmembrane domain 1 of GABAA receptors, which were obviously important for allosteric modulation or direct activation of GABAA receptors by steroids, respectively (Hosie, Wilkins, da Silva, & Smart, 2006). Using a homology model based on the structure of the transmembrane domain of the nACh receptor (Miyazawa, Fujiyoshi, & Unwin, 2003), the identified residues were assigned to opposite faces of the α1M1 helix, strongly suggesting that they contribute to two distinct binding sites within the transmembrane domain of GABAA receptors. Further considerations and mutations based on their model structure identified additional amino acid residues possibly contributing to two binding sites: one site with contributions of residues α1Gln241 and α1Asn407 seemed to be located within the four transmembrane helices of the α subunit, mediating allosteric modulation of receptors at low steroid concentrations. Another binding site with contributions of residues, α1Thr236 and β2Tyr284, seemed to be located at the β+α  interface below the GABA-binding pocket in the transmembrane domain and mediates direct activation of GABAA receptors at high steroid concentrations (Hosie et al., 2006). In addition to these steroid-binding sites, other binding sites might exist for the inhibitory sulfated steroids (Hosie, Wilkins, & Smart, 2007).

6.6. A possible loreclezole-binding site near β2Asn265 at the extracellular end of TM2 The GABA-enhancing action of the anticonvulsant loreclezole depends on the presence of a β2 or β3 subunit in GABAA α1βγ2 and α1β receptors, being approximately 300-fold weaker in receptors containing β1 subunits (Wafford et al., 1994). Site-directed mutagenesis studies led to the

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identification of a single amino acid residue in the β2 and β3 subunit, that is located at the carboxy-terminal (extracellular) end of TM2 (TM2 150 ; Fig. 1B and C). Thus, the mutation β2Asn289Ser or β3Asn290Ser eliminated loreclezole sensitivity from β2- or β3- containing receptors, respectively (Wingrove et al., 1994), and the mutation β1Ser290Asn conferred loreclezole sensitivity to β1-containing receptors (the amino acid numbering here comprises the signal peptide and corresponds to β2/β3Asn265Ser in the mature subunit). A similar dependence on these amino acid residues in β2 or β3 subunits was also observed for the GABA-enhancing actions of etomidate (Belelli et al., 1997), of the anxiolytic and anticonvulsant tracazolate (Thompson, Wingrove, Connelly, Whiting, & Wafford, 2002), of some nonsteroidal anti-inflammatics such as mefenamic acid (Halliwell et al., 1999; Smith, Oxley, Malpas, Pillai, & Simpson, 2004), of some γ-butyrolactones (El Hadri et al., 2002), as well as of some flavan-3-ol compounds (Fernandez et al., 2012). This amino acid residue also seems to be important for the positive GABAA receptor-modulatory action of high concentrations of some β-carbolines (DMCM and others; Section 4.2.1) (Stevenson et al., 1995; Thomet et al., 1999), or for the inhibitory action of furosemide at α6β2/3γ2 receptors (Korpi, Kuner, Seeburg, & Luddens, 1995; Thompson et al., 1999). Interestingly, a newly identified flavan Fa173 (cis-(2S,3S)-3acetoxy-30 ,40 -dimethoxyflavan) antagonized not only the potentiating actions of the flavan-3-ol Fa131 (trans-(2S,3R)-3-acetoxy-40 methoxyflavan; Fernandez et al., 2012) but also that of etomidate, loreclezole, and of high concentrations of diazepam (see Section 4.2.1) at α1β2 and α1β2γ2L GABAA receptors. The action of this antagonist thus suggests that all these compounds might interact with the same “loreclezole”-binding site. Fa173, however, did not antagonize the potentiation induced by propofol, the neurosteroid (3α,5β)-3-hydroxy-pregnan20-one, or the barbiturate thiopental (Fernandez et al., 2012), supporting the conclusion that the latter compounds seem to bind to a different site. Mutagenesis studies in transmembrane domains, however, generally are difficult to interpret, because amino acid residues in the transmembrane domain apparently important for drug action might either be part of the respective binding site or be important for the transduction of drug effects. The observation that the action of multiple and structurally different drugs depends on a single residue rather argues for an involvement of the residue β2Asn265 in the transduction mechanism and not in the binding of these drugs. This conclusion is supported by the observation that mutation of this


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residue dramatically reduces the efficacy of etomidate for enhancing GABAinduced currents, but not its binding affinity (Desai, Ruesch, & Forman, 2009). In addition, in structural models of the GABAA receptor transmembrane domains based on disulfide cross-linking and photolabeling data, β2Asn265 is located outside the intersubunit cleft where etomidate binds (Desai et al., 2009).

6.7. A possible n-octanol-binding site near β2Asn265 These data, however, do not rule out that this residue does not also contribute to a ligand-binding site. Using a cysteine substitution of β2Asn265, it was demonstrated that propofol could not protect this cysteine from being modified by a sulfhydryl-specific reagent (Bali & Akabas, 2004), but n-octanol does (McCracken, Borghese, Trudell, & Harris, 2010). These and other results led to the conclusion that β2Asn265 might be close to a binding site for alcohol and volatile anesthetics (McCracken et al., 2010). Thus, this residue may contact modulators in intersubunit sites, as observed with ivermectin in GluCl (Hibbs & Gouaux, 2011) and small alcohols and bromoform in positively modulated GLIC mutants (Bromstrup et al., 2013). Data from mutagenesis studies thus have to be supported by data obtained with other techniques and ligand-induced protection against cysteine modification is one of the methods providing additional information.

6.8. Conclusions on the localization of anesthetic binding sites in GABAA receptors 6.8.1 Possible pitfalls of the methods used for localization of anesthetic binding sites Some of the discrepancies between early mutagenesis work and later results might have been caused by the fact that loss of a drug effect by mutagenesis can have several reasons and cannot distinguish between residues directly involved in binding or in the transduction of an effect. Elimination of drug action via covalent modification of introduced cysteines by sulfhydrylspecific reagents, or covalent labeling of amino acid residues using photoreactive ligands, seems to be more suitable for the identification of residues contributing to a binding site, especially if the chemical reaction can be blocked by the ligand in question. Results obtained with the photolabeling technique, however, might depend on the structure of the photoreactive ligand and its reactivity toward different types of amino acid residues, thus possibly explaining some of the discrepant results obtained so far. In addition, due to the low efficacy of

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photolabeling and the methodological difficulty of identifying labeled residues from small amounts of proteins, some of the residues that theoretically could have been photolabeled might have been overlooked. In addition, results obtained might depend on the lifetime of the photoreactive intermediate and possible structural rearrangements within the ligand. Nevertheless, photolabeling experiments combined with competitive inhibition of the photoreaction by the respective ligand seem to be one of the strongest strategies for the localization of ligand-binding sites. Other discrepancies might have been caused by differences in assigning amino acid residues to the intra- or intersubunit-binding pockets resulting from the use of different structural models of the GABAA receptor transmembrane domain. However, the structural models used so far have now to be compared with the crystal structure of the homo-oligomeric β3 GABAA receptor (Miller & Aricescu, 2014) to clarify whether they reliably depicted the situation in GABAA receptors. In the structure of the β3 receptor transmembrane domain, there are fundamental differences as compared to nACh receptors, GLIC, ELIC, or GluCl. In addition, the propofolbinding pocket identified in the bacterial channel GLIC (Nury et al., 2011) is structurally distinct from the respective pocket within the β3 receptor. Similarly, the intersubunit interfaces from currently available crystal structures of ligand-gated ion channels reveal considerable differences in the geometry and thermodynamics of complex formation, with GABAA receptor β3 crystal subunits forming the most extensive and energetically favorable interactions (Miller & Aricescu, 2014). It is quite clear that these differences also will influence the pocket structure and conformational changes induced in the receptor (see below). And currently, it cannot be predicted whether this β3 crystal structure can be used as a reliable template for all possible hetero-oligomeric GABAA receptors. Similarly, cysteine-cross-linking experiments not necessarily provide correct information on the relative orientation of the transmembrane helices of GABAA receptors because any mutation within the transmembrane domain could cause and cross-linking could prevent conformational changes that might influence the results of the experiments. A slightly different conformation of the transmembrane helix to which the respective amino acid residue contributes could result in its pointing into the intrasubunit or the intersubunit pocket. In addition, the exact movements and possible rotations of the transmembrane helices during opening of the channel or allosteric modulation of the receptor by ligands are not known, again making assignment of amino acid residues to the one or the other pocket type difficult.


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Finally, the existence of linking channels between intersubunit and intrasubunit pockets identified in GLIC (Nury et al., 2011), and the lateral tunnels running between subunits in the extracellular domain into the central vestibule of β3 receptors (Miller & Aricescu, 2014) together with the high flexibility of TM3 indicate that the respective structures may not form well-defined pockets. Pockets may either communicate or even fuse with each other or disappear in certain conformational states of the protein. This dynamical view could well resolve some of the discrepancies concerning anesthetic binding sites (Nury et al., 2011). Localization of drug-binding sites by crystal structures of ligand-bound receptors in principle is the most powerful technique because it not only provides direct information on the location of the drug-binding sites without having to use model structures but also provides information on the concomitant conformation of the receptors. Interestingly, the crystal structure of the GABAA receptor homo-oligomeric β3 receptor seems to be in the desensitized state (Miller & Aricescu, 2014). In the absence of additional crystal structures of this receptor in the resting and open state, however, no conclusion on possible ligand-induced conformational changes can be made. Some drugs, such as picrotoxinin (Korshoej et al., 2010) or some anesthetics (Willenbring, Liu, Mowrey, Xu, & Tang, 2011), seem to be able to induce or stabilize desensitized states. To clarify the conformational changes occurring within GABAA receptors during activation, allosteric modulation, and desensitization, multiple crystal structures of GABAA receptors in the absence or presence of various bound ligands have to be generated and compared with each other. However, crystal structures also have their pitfalls, such as providing a frozen picture of receptor and ligand with unclear state of the receptor (open, closed, and desensitized), and the possible presence of detergents or lipids within the receptor that could potentially block binding sites or cause a rearrangement of some parts of the molecule (Nury et al., 2011). In addition, possible changes in the structure of the receptors during solubilization, purification, and crystallization under conditions not comparable with conditions in vivo cannot be excluded. So all available methods have to be applied and their results have to be compared to come to a consensus localization and structure of a ligand-binding site. But in any case, it has to be stressed that crystal structures of GABAA receptors and not of homologous proteins have to be investigated to obtain relevant structural information on these receptors. Ligand-bound crystal structures from GLIC, ELIC, or GluCl confirm the existence of binding sites

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within the various pockets and indicate where such binding sites might be in GABAA receptors. But due to the difference in amino acid residues forming the respective binding sites of homologous proteins, the respective pocket size and shape could be different and could influence the exact location of the binding site. 6.8.2 Summary on the localization of binding sites of various anesthetics As indicated in the introduction of Section 6 experiments performed during the last 17 years confirmed that anesthetics interact with more than one binding site in GABAA receptors and that these sites are located in the transmembrane domain. Further experiments will have to clarify whether additional binding sites for anesthetics can also be found outside of the transmembrane domain of GABAA receptors, as suggested by the cocrystal structure of ELIC and bromoform (Spurny et al., 2013). The proposal that inhalation anesthetics seem to bind to the intrasubunit pocket in α subunits and intravenous anesthetics to the homologous intrasubunit pocket in β subunits no longer seems to hold true for all these drugs. This conclusion is supported by the now amply confirmed finding that etomidate binds to the β+α  transmembrane interface (below the GABA-binding site) and that at least one inhalation anesthetic seems to competitively interact with the etomidate site (Li et al., 2006). The binding sites of barbiturates seem to be located at the α+β  and β+γ  interfaces, but obviously, structural analogues of etomidate and barbiturates might partially act via all three types of interfaces, and the same probably is the case with propofol. The existence of multiple and partially or fully shared binding sites of these compounds in the transmembrane domain, as well as conformational changes induced in other pockets by binding of these ligands (see Section 6.3.1), explains the difficulty of clearly identifying a competitive interaction of these compounds. Thus, mutual inhibition of these compounds is partially allosteric and partially competitive, although these compounds might interact formally with the same binding sites. There are, however, still some open questions. Thus, the site of action of inhalation anesthetics currently is not clear, with the possible exception of the enflurane-binding site, that might be identical to the etomidate-binding site. Further experiments are required to confirm this conclusion and to locate the site of action of other inhalation anesthetics. There also seems to be consensus that steroids seem not to interact with the etomidate-, barbiturate-, and propofol-binding sites, and the


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steroid-binding sites claimed to be at the β+α transmembrane interface and within the intrasubunit pocket of α subunits so far have not been confirmed. Studies using suitable steroid photolabels in the absence or presence of steroids and other compounds might provide additional and more detailed information. A recently identified novel steroid-binding site in TM3 of the homo-oligomeric β3 GABAA receptors (Chen et al., 2012) is just the starting point of more detailed similar studies in hetero-oligomeric receptors. Similarly, the localization of the loreclezole-binding site currently is not known, given the discussion in Section 6.6 on the probability that residue β2Asn265 in the transmembrane domain with high probability might be a transduction site. The possibility that this residue might also contribute to a binding site has to be further investigated by characterizing possible ligands for this site.

7. ALCOHOL-BINDING SITES 7.1. Alcohol-binding sites in the transmembrane domain Ethanol and longer chain alcohols exhibit a multiplicity of actions at various receptors and proteins. Nevertheless, the potencies of the n-alcohol series for physiologic immobilization and anesthesia parallel that for the potentiation of native GABA-induced currents (Nakahiro, Arakawa, Nishimura, & Narahashi, 1996), suggesting that GABAA receptors are an important site of action of alcohols. Over the time, several alcohol-binding sites have been identified in GABAA receptors, and depending on the receptor type in which they were investigated, they enhanced or reduced GABA-induced currents (for review, see Howard et al., 2014). Alcohols can act on GABAA receptors at sites defined by transmembrane amino acid residues also crucial for the actions of anesthetics like etomidate and propofol (Mihic et al., 1997), as well as the amino acid residue important for loreclezole action (McCracken et al., 2010). In addition, the general anesthetic sites also appear to be related to the ethanol sites identified in the crystal structures of an ethanol-sensitized GLIC variant. Both are located in a transmembrane cavity between channel subunits (Sauguet et al., 2013) and may stabilize the open form of the channel. These effects, however, require very high concentrations of ethanol (of 100 mM or higher) (Wallner et al., 2014). In contrast, pronounced ethanol effects are observed already at doses at and below 10 mM (the blood-alcohol driving limit in most EU countries, which is related to impaired human neurobehavioral functions).

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7.2. Alcohol-binding sites in the extracellular α+β 2 interface of α4/6β3δ receptors The finding that the imidazobenzodiazepine Ro15-4513 (a high-affinity ligand for the benzodiazepine-binding site of GABAA receptors) was able to inhibit at least some of the effects of lower doses of acute ethanol (Suzdak et al., 1986), as well as the identification of an α6(R100Q) polymorphism in an alcohol nontolerant rat line, that enhanced the ethanol sensitivity of mice (Korpi, Kleingoor, Kettenmann, & Seeburg, 1993), provided the first hints for an extracellular location of an ethanol site. Later on, it was shown that the increased motor-impairing effects of ethanol in alcoholnontolerant α6R100Q rats can be explained by increased alcohol sensitivity of tonic currents in cerebellar granule cells and that the α6R100Q mutation further increases the already high alcohol sensitivity of α4/6βδ receptors, but only when these receptors contained a β3 subunit (Hanchar et al., 2006). This conclusion was supported by the demonstration of a high-affinity [3H]Ro15-4513 binding site in the extracellular domain of α4/6β3δ receptors that could be blocked by low concentrations of ethanol (Hanchar et al., 2006). Mutagenesis studies indicated that the β3 ethanol selectivity is determined by a single amino acid residue (β3Y66) of the extracellular domain of the β3  side that differs between different GABAA receptor β subunits. This residue is located opposite to the α6R100 benzodiazepine-binding site residue and this ethanol-binding site is thus located at the α6+β3 interface (Section 4.2.2) at a position homologous to that of the classical benzodiazepine-binding site at the α6+γ2  interface. This site not only binds ethanol at physiologically relevant concentrations (EC50 ¼ 17 mM) but also exhibits a high affinity for some selected benzodiazepine site ligands, such as the alcohol antagonistic imidazobenzodiazepine Ro15-4513 and others (Wallner et al., 2014). It remains to be determined, however, which active sites in which receptor subtypes are critical for the various actions of alcohols or whether a dynamic balance of multiple sites underlies their clinical phenotype (Howard et al., 2014). Interestingly, this high-affinity binding site for Ro15-4513 and ethanol not only depends on the α4/α6 and β3 subunits, that form the binding site, but is also dependent on the presence of a δ subunit within the receptor (Wallner et al., 2014). A similar dependence on a subunit not directly involved in binding of the ligand was also observed for GABA (Baumann et al., 2003; Ducic, Caruncho, Zhu, Vicini, & Costa, 1995; Ebert, Wafford, Whiting, Krogsgaard-Larsen, & Kemp, 1994), neurosteroids


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(Bianchi & Macdonald, 2003), the nonsedative anxiolytic agent tracazolate (Zheleznova, Sedelnikova, & Weiss, 2008), or the pyrazoloquinolinones (Mirheydari et al., 2014). So far, this has not been observed for the classical benzodiazepine-binding site at the homologous α+γ interface, and thus, the effects of benzodiazepines are assumed to be independent of the type of β subunit present in the receptor. However, only a few sets of data are available on the influence of the β subunit type on the efficacy of benzodiazepines and even in these data some small efficacy differences between receptors containing different β subunit types were observed (Hadingham et al., 1993). A much more extensive characterization of various benzodiazepine site ligands presumably will indicate a much stronger influence of the type of β subunit on the allosteric modulation by some benzodiazepines. The effects of a subunit apparently not involved in binding of the ligand for sure are caused by an allosteric interaction of this subunit with the subunits forming the binding site. Whether this allosteric interaction is caused by the structure and flexibility of this “third” subunit type alone, or is elicited by an additional interaction of the ligand with a so far unidentified binding site at this subunit, will have to be investigated in the future.

8. CANNABINOID-BINDING SITE The endocannabinoid system is part of a complex lipid signaling network involving the G protein-coupled receptors CB1 and CB2. Several lines of evidence indicate that endocannabinoids are also involved in the regulation of GABA and glutamate release (Rea, Roche, & Finn, 2007). In addition, recently, it was demonstrated that 2-arachidonyl glycerol and other endocannabinoids are able to directly modulate GABAA receptors via a novel-binding site located in the TM4 domain of GABAA receptors containing a β2 subunit (Sigel et al., 2011). Cysteine scanning mutagenesis then indicated that the endocannabinoid-binding site is located between TM3 and ˚, TM4 of the β2 subunit and that the important residues span >18 A suggesting a near linear conformation of the 2-arachidonyl glycerol, and indicating that the site mainly locates to the inner leaflet of TM4 and stretches far into the membrane. This conclusion is convincingly supported by docking experiments of 2-arachidonyl glycerol into a structural model of a GABAA receptor (Baur et al., 2013). This site thus suggests additional possibilities for binding of ligands that so far have not been considered. Similar binding sites located between TM3 and TM4, or other TMs, might also be available at other subunits for other compounds interacting with GABAA receptors.

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9. AVERMECTIN B1a-BINDING SITE Avermectin B1a, an anthelmintic macrocyclic lactone, is widely used as an antiparasitic agent in domestic animals, usually as a mixture (ivermectin) of avermectin B1a and avermectin B1b. The target of its antiparasitic action is believed to be an ivermectin-sensitive glutamate-gated Cl channel that is directly activated by ivermectin at nanomolar concentrations and that is found exclusively in invertebrates (Lynagh & Lynch, 2012). At higher (micromolar) concentrations, avermectin B1a also directly activates vertebrate Cys-loop receptors such as nACh, GABAA, and glycine receptors. Activation of these receptors is much slower than that induced by GABA and the slow desensitization of current was sometimes interpreted as an irreversible action of avermectin. However, due to its lipophilic nature, it is likely that avermectin B1a accumulates in the membrane and binds reversibly and weakly to its site (Lynagh & Lynch, 2012), thus constantly activating the receptor. At GABAA and glycine receptors, however, there are also some effects of avermectin B1a at low concentrations. Thus, avermectin B1a exhibits a high-affinity binding site in brain membranes that is associated with GABAA receptors (Drexler & Sieghart, 1984a, 1984b, 1984c). At low concentration, avermectin B1a enhances GABA or glycine-activated current (Shan, Haddrill, & Lynch, 2001; Sigel & Baur, 1987), whereas at higher concentrations, it directly activates these channels, and Hill coefficients indicate that at least two binding sites have to cooperate for direct activation (Adelsberger, Lepier, & Dudel, 2000; Shan et al., 2001). The crystal structure of GluCl complexed with ivermectin for the first time visualized the location of the directly activating ivermectin-binding site in a Cys-loop receptor (Hibbs & Gouaux, 2011) and indicated that ivermectin is binding in the transmembrane domain in a cleft between TM3 of one subunit (+ side) and the M1 of an adjacent subunit ( side) at the GluCl receptor (Fig. 1B). By probing ivermectin sensitivity determinants on the α1 glycine receptor using site-directed mutagenesis and electrophysiology (Lynagh, Webb, Dixon, Cromer, & Lynch, 2011), as well as by performing molecular modeling of a putative ivermectin-binding site using ELIC as a template, a glycine receptor ivermectin-binding orientation similar to that in GluCl was suggested. Nevertheless, some of the binding interactions revealed by this GluCl structure do not pertain to other highly ivermectinsensitive cys-loop receptor. This conclusion is supported by data discussed in Lynagh and Lynch (2012), for GABAA and nACh receptors. Thus,


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computer docking simulations from two laboratories indicated that the lowest energy docking of ivermectin to the α7 nACh receptor was in an intrasubunit cavity as opposed to the interfacial site in anionic receptors. In both studies, the ivermectin molecule wedges between M1 and M4 from the same subunit, partly in contact with the surrounding lipids, with the benzofuran moiety oriented toward either M2 or M3 of the same subunit. The directly activating avermectin B1a-binding site at GABAA receptors also cannot be defined from the GluCl–ivermectin crystal structure (Lynagh & Lynch, 2012). In addition, the structural basis for the highaffinity binding and modulation by avermectin B1a of certain GABAA and nACh receptors so far has also not been identified.

10. BINDING SITES OF IONS In addition to the modulation by multiple drugs, GABAA receptors are also modulated by various cations, such as protons (Huang, Chen, & Dillon, 2004; Wilkins, Hosie, & Smart, 2002), Zn2+ (Fisher & Macdonald, 1998; Horenstein & Akabas, 1998; Hosie, Dunne, Harvey, & Smart, 2003), La3+ (Zhu, Wang, Corsi, & Vicini, 1998), or Cu2+ (McGee, Houston, & Brickley, 2013). Again, multiple binding sites for most of these ions have been identified by site-directed mutagenesis that are located either within the ion channel or within the extracellular domain. Since these binding sites in many cases are formed by specific amino acid residues, they differ in distinct receptor subtypes.

11. CONCLUSION From the previous chapters, it is clear that most, if not all, GABAA receptor ligands can interact with more than one binding site at these receptors. Most of these binding sites are not exactly defined yet and it can be assumed that identification of these sites will be an extremely difficult task given the high flexibility of the receptors and the existence of multiple binding sites. Since binding of a ligand in most cases causes changes in the structure of the receptor, a second ligand with comparable affinity for the same site can be redirected to another site and allosterically and not competitively influence the binding of the first ligand. Such mechanisms can contribute to the difficulty in exactly defining the sites of action of various anesthetics that all are modulated by similar amino acid residues (see Section 6). As discussed there, photoaffinity labeling of the binding

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sites with and without protection by ligands and the generation of ligandbound crystal structures of GABAA receptors seem to be suitable techniques for identifying these sites and their location. To speed up the generation of data from ligand-bound crystal structures of GABAA receptors, electron crystallography might be considered that avoids the generation of large crystals, a bottleneck of X-ray crystallography (Shi, Nannenga, Iadanza, & Gonen, 2013). In addition, it has to be stressed that the various sites of interaction of a single ligand might be different in different receptor subtypes. Differences in the amino acid sequences of the subunits for sure cause differences in the size and shape of the binding sites at the interfaces or pockets of a receptor, thus allowing drugs to bind or not to bind. The presence or absence of modulation via such binding sites for sure will influence the receptor subtype pharmacology of this ligand. Differences in the distribution of hydrophobic and hydrophilic regions and altered possibilities for hydrogen bonds or cation–π interactions might not only change the affinity of a ligand for different receptor subtypes, but possibly also their orientation within the pockets. In addition, these structural differences presumably also allow different drug-induced movements and a different signal transduction of the receptors, thus influencing their efficacy for modulation of GABA-induced currents. In addition, affinity and efficacy of compounds are modulated by all subunits present within the receptor and this is independent of the location of the respective binding sites, as discussed in Section 7. All these factors result in the sometimes extreme differences in binding affinity and efficacy of ligands in different receptor (Rudolph & Knoflach, 2011) subtypes, that is found throughout the various GABAA receptor-binding sites (e.g., binding sites for GABA (Karim et al., 2013; Mortensen, Patel, & Smart, 2011), for benzodiazepines (Knust et al., 2009; Sternfeld et al., 2004), for pyrazoloquinolinones (Mirheydari et al., 2014; Varagic, Ramerstorfer, et al., 2013; Varagic, Wimmer, et al., 2013), for neurosteroids (Bianchi & Macdonald, 2003), or for tracazolate (Zheleznova et al., 2008)). In addition, such differences in the binding pockets of different receptor subtypes might change the allosteric modulation of drugs from null modulator to positive or negative allosteric modulator, as for instance with flumazenil that is a null modulator at α1β3γ2 and α5β3γ2 receptors, but a positive allosteric modulator at α2β3γ2, α3β3γ2, α4β3γ2, and α6β3γ2 receptors (Ramerstorfer, Furtmuller, Vogel, Huck, & Sieghart, 2010). And of course, a different amino acid sequence of a subunit might allow novel interactions with drugs that are not observed


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in other receptor subtypes (e.g., with furosemide (Korpi et al., 1995) or with Ro15-4513 and ethanol (Wallner et al., 2014)). From all that it is clear that for clarification of the spectrum of action of a compound, its interaction with each individual receptor subtype has to be investigated. This requires the investigation of multiple receptor subtypes. Thus, to completely characterize all γ2-containing receptors, the investigation of 18 different receptor subtypes (six α  three β  one γ2) will be necessary with a single ligand. A first screening can be performed using a medium and a high concentration of the compound at each receptor subtype. If the compound is able to significantly induce or modulate currents in the absence or presence of GABA, respectively, complete concentration–response curves have to be performed and also to be published. Investigation of multiple receptors is especially important if a possible receptor subtype selectivity of a compound is claimed. Only then it can be clearly delineated at which concentration the compound is selectively modulating a single receptor subtype and to which extent the compound is modulating additional receptor subtypes at the concentrations used. Unfortunately, such a detailed analysis is extremely rare in the literature. In most cases, only two or three receptor subtypes have been investigated and only the EC50 and the maximum modulation at high compound concentrations is given. Such data might be sufficient for a preliminary characterization of compounds, but are not helpful for estimating the selectivity of a compound for a receptor subtype. In addition, it is not useful to present the data relative to that of another compound, for instance to present the action of a novel benzodiazepine site ligand relative to that of chlordiazepoxide, as often done in the literature. Although this type of data shows whether a compound is more or less efficacious than chlordiazepoxide at the various receptor subtypes, in the absence of concentration–response curves it does not allow to account for a possible difference in the concentration dependence of these two compounds. Furthermore, even when relative concentration–response curves are given, in the absence of the concentration–response curve of chlordiazepoxide used for this comparison, these data cannot be compared with those of other compounds. So this type of data conveys only part of the information available and is not suitable for a complete and helpful information transfer to the public. Although results in the last couple of years have indicated that the pharmacology of GABAA receptors is much more complex than previously assumed, they also have identified ligands that are highly selective for certain receptor subtypes (Dias et al., 2005; Sternfeld et al., 2004). In addition, by

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exploiting the interaction of ligands with other binding sites, such as those at the α+β  interface (Varagic, Ramerstorfer, et al., 2013; Wallner et al., 2014), there is ample room for the development of receptor subtypeselective compounds and it can be expected that in the near future we will have a complete set of compounds that can modulate any one of the 18 γ2containing receptors with high selectivity. That will not only boost basic science by clarifying the role of individual receptor subtypes in different behavior but will also open new avenues for the treatment of various diseases (Rudolph & Knoflach, 2011; Rudolph & Mohler, 2014).

CONFLICT OF INTEREST The author has no conflicts of interest to declare.

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Werner Sieghart

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Allosteric modulation of GABAA receptors via multiple drug-binding sites.

GABAA receptors are ligand-gated ion channels composed of five subunits that can be opened by GABA and be modulated by multiple pharmacologically and ...
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