7 GABAA receptors: Structure, function and modulation

Research Signpost 37/661 (2), Fort P.O., Trivandrum-695 023, Kerala, India

Biological and Biophysical Aspects of Ligand-Gated Ion Channel Receptor Superfamilies, 2006: ISBN: 81-7736-254-2 Editor: Hugo R. Arias

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GABAA receptors: Structure, function and modulation Ren-Qi Huang, Eric B. Gonzales and Glenn H. Dillon Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, TX 76107, USA

Abstract The GABAA receptor (GABAAR) is the predominant inhibitory neurotransmitter in the mammalian central nervous system, and is the site of action of numerous therapeutic agents. This receptor is part of a superfamily of ligand-gated ion channels, which also include receptors for acetylcholine (nicotinic), glycine and serotonin (5-HT3 subtype). The first GABAAR subunits were cloned in 1987; since then, at least 18 additional subunits have been cloned. Advances in molecular biology and structural analysis, combined with biochemical and electrophysiological techniques, have led to significant advances in our understanding of the physiological and pharmacological properties of this important class of neurotransmitter receptors. This review summarizes our current understanding of the structure and function of the GABAAR, as well as pharmacologic and physiologic mechanisms by which its activity is modulated. Correspondence/Reprint request: Dr. Glenn H. Dillon, Dept. of Pharmacology and Neuroscience, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107, USA. E-mail: gdillon@ hsc.unt.edu

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Abbreviations 5α3α-THPROG, 5α-pregnan-3α-ol-20-one; 5-HT3R, 5-hydroxytryptamine type 3 receptor, ACh, acetylcholine; nAChR, nicotinic acetylcholine receptor; AChBP, acetylcholine binding protein; BZD, benzodiazepine; CNS, central nervous system; FTZ, flunitrazepam; GABA, γ-aminobutyric acid; GABAAR, γ-aminobutyric acid type A receptor; GABABR, γ-aminobutyric acid type B receptor; GABACR, γ-aminobutyric acid type C receptor; Gly, glycine; GlyR, glycine receptor; LGIC, ligand-gated ion channel; PTX, picrotoxin; SCAM, substituted cysteine accessibility method; TBPS, tbutylbicyclophosphorothionate; PKC, protein kinase C.

1. Introduction

γ-aminobutyric acid (GABA) is the predominant inhibitory neurotransmitter in the mammalian central nervous system (CNS). Its effects are mediated via its interaction with two major structural classes of receptors. Type B GABA receptors (GABABRs) belong to the family of G protein-coupled receptors, and mediate their effects through activation of several signal transduction pathways (Couve et al., 2000). GABAARs, the better characterized and more widespread of the receptor classes, belong to a superfamily of ligand-gated ion channels (LGICs). A third type of GABAAR, prominent in the retina, but also present in some brain regions, shares significant structural homology with the GABAAR. Because of its unique pharmacology and distinct channel characteristics, however, it has been termed the GABAC receptor (GABACR) (see Bormann, 2000). GABAARs are the focus of this chapter. Binding of GABA (in most cases probably two molecules) to the GABAARs results in opening of a Cl- channel. The direction of movement of Cl- ions through this channel depends upon the relative concentration of intra- to extracellular Cl-. In the adult CNS, the extracellular concentration of Cl- ([Cl-]o) is typically higher than the intracellular concentration ([Cl-]i), and thus Cl- moves into the cell. This causes hyperpolarization of the neuron, and consequently neuronal activity is reduced. The net physiologic effect to the brain of GABAAR activation, then, is an inhibition of brain activity. Excitatory stimuli also impinge upon most neurons in the brain. The regulated interplay between these excitatory and inhibitory inputs maintains normal brain function. When an imbalance between these opposing systems occurs, abnormal at worst, or, at the minimum, undesirable brain function results. With regards to the inhibitory aspects of brain function, alterations in GABAergic neurotransmission have been implicated in severe CNS disorders. For instance, a number of mutations found in GABAARs cause several forms of epilepsy (recently reviewed by Macdonald et al., 2004). In addition, work in recent years has established a strong link between GABAergic signaling and schizophrenia (see Caruncho et al., 2004; Costa et al., 2004). Moreover, the role of GABAARs in anxiety and related disorders is well known (Nemeroff, 2003). Because of this direct involvement in several disorders, as well as the ubiquitous nature of the GABAARs, it continues to be a target for therapeutic intervention. Thus, a thorough understanding of the operation of this receptor is vital for continued advances in CNS drug development. Increased technical capabilities in recent years have made it possible to gain insight into the GABAAR and its key functional domains. This chapter will focus on advances in our

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understanding of the structure and critical functional domains of the receptor. In addition, mechanisms through which the activity of the receptor is modulated will be reviewed.

2. Structural features of the GABAA receptor 2.1. Multi-subunit, pentameric structure

The GABAAR is a receptor/Cl- channel complex, and is composed of several distinct subunits. The first of these subunits was cloned in the late 1980’s (Schofield et al., 1987; Lynch et al., 1995; 1997; Dibas et al., 2002); since then, fifteen additional subunits have been cloned. These subunits have been assigned to classes α, β, γ, δ, ρ, ε, π, and θ (see Lynch et al., 1995; 1997; Siegwart et al., 2002). Multiple isoforms exist for many subunits; these are so designated due to amino acid sequence homology. For instance, subunit isoforms of the α class share approximately 70-80% homology with other α subunits, but only approximately 25-40% homology with β or γ subunits (and only 1520% homologous with subunits from different members of the superfamily). In addition, several isoforms also demonstrate splice variants e.g., γ2short (γ2S) and γ2long (γ2L) (Whiting et al., 1990), and β2short (β2S) and β2long (β2L) (McKinley et al., 1995). The individual subunits that form the GABAAR average roughly 50 kDa in size (ranging from approximately 450-550 amino acids in length). The general structure of the GABAAR is similar to that of several other receptor/channel complexes, which together form the superfamily of Cys-loop LGICs. This superfamily includes nicotinic acetylcholine receptors (nAChRs), glycine receptors (GlyRs), and serotonin type 3 receptors (5-HT3Rs). Analysis of hydropathy plots indicates that each subunit has four hydrophobic transmembrane domains (TM1-TM4). A large intracellular loop, containing consensus sites for phosphorylation, exists between TM3 and TM4 in several subunits. TM2 is the most highly conserved domain of LGICs, and forms the central ion pore. Whereas a crystal structure has not been obtained for any of these proteins, the recent crystallization of the acetylcholine binding protein (AChBP) (Brejc, van Dijk et al. 2001) has provided an exceptional template to model the extracellular aspect of Cysloop LGICs (described below). Early electron microscopy studies demonstrated that these proteins consist of five subunits, arranged pseudosymmetrically around a centralized pore (Toyoshima and Unwin, 1988; Nayeem et al., 1994). Since all Cys-loop LGICs share a conserved architecture, data derived from one receptor has reliably served as a template for the others in the family. Until the crystallization of the AChBP, studies on nAChRs from Torpedo californica provided the bulk of structural information about Cys-loop LGICs. These receptors have been extensively imaged by Unwin and colleagues, who have utilized computer-enhanced electron microscopic analysis to determine the structure of the protein to a resolution of 4.6 Å (Miyazawa et al., 1999). Most recently, the structure was improved to 4 Å (Unwin, 2005). Results from these investigators demonstrate the transmembrane domains are α-helical, and they suggest the presence of a constricting hydrophobic region near the midpoint of TM2, which may serve as the channel gate. Additional techniques, such as the substituted cysteine accessibility method (SCAM), Karlin and colleagues (1992) have also provided critical information about structure of the receptor transmembrane domains. Xu and Akabas (1996) used the SCAM method to identify channel-lining residues in the a1 subunit TM2

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domain. Using α1β1γ2 GABAARs expressed in Xenopus oocytes, the authors were able to confirm that the TM2 domain is α-helical in structure. Another technique that has provided insight into the channel structure is the tryptophan scanning method. The TM3 and TM4 domains have been the focus of tryptophan scanning in both nAChRs and GABAARs (Cruz-Martin et al. 2001; Jenkins et al., 2002). In the TM4 domain of GABAARs, tryptophan scanning revealed that changes in agonist sensitivity upon tryptophan introduction followed the periodicity of an α-helix (Jenkins et al., 2002). Results using various biochemical approaches also indicate the predominant structure of the transmembrane helices of receptors in the Cys-loop LGIC superfamily is a helix (Barrantes et al., 2000; Methot et al., 2001). Thus, there is general agreement that the TM domains of the ligand-gated ion channels are predominantly α-helical. However, it should be noted that the presence of some non-helical regions, particularly in TM1, may exist (Leite and Cascio, 2001).

2.2. Stoichiometry of the GABAA receptor

It is generally accepted that the adult form of nicotinic receptor of skeletal muscle is composed of two α subunits, one β subunit, one ε subunit (the ε subunit is replaced by γ in the fetal form of the receptor), and one δ subunit (Lindstrom, 2003). In contrast, the stoichiometry of the GABAAR is less widely agreed upon. Whereas it is agreed that the predominant form of the native GABAAR incorporates α, β, and γ subunits, the stoichiometry of these subunits has been a question of debate. Immunoprecipitation studies of native GABAARs, using subunit specific antibodies, have indicated that two α subunits exist in a single receptor (Pollard et al., 1993; Pollard et al., 1995; Khan et al., 1996). Although the population of receptors possessing two isoforms of the α subunit was minor, it suggests that the likely structure of the native receptor is one which incorporates two α subunits, which may or may not be the same isoform. Patch clamp analysis of cells transfected with multiple α subunits, in conjunction with a β and γ subunit, also indicated a functional receptor using both α subunits formed (Verdoorn, 1994). All of these results suggest the native receptor likely utilizes two α subunits, which may or may not be identical. Results from immunoprecipitation analysis suggested that individual receptors may also possess two different isoforms of the γ subunit (Quirk et al., 1994), indicating the native receptor may also incorporate two γ subunits. According to this view, the native receptor would be composed of 2 α subunits, 2 γ subunits, and a single β subunit. Although this configuration has not been definitively dismissed, the majority of evidence in more recent years indicates a stoichiometric arrangement of 2 α, 2 β, and 1 γ subunit. Im et al. (1995) constructed a tandem construct of α6 and β2 subunits. The tandem construct, either alone or in combination with α6, β2, or γ2 subunits, was transfected into HEK293 cells, and analyzed through both binding and patch clamp studies. High affinity binding sites for the GABA agonist muscimol were formed in cells transfected with any combination of receptor subunits, including the tandem construct alone. However, the expression of functional GABA-gated channels could only be detected in those cells that were also transfected with either a γ2 or α6 subunit, in addition to the α6β2 tandem construct. The results led Im et al. (1995) to conclude that GABAARs

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composed of three subunits form using 2 α subunits, 2 β subunits, and 1 γ subunit. Subsequent studies by several other laboratories support this model (Chang et al., 1996; Tretter et al., 1997; Farrar et al., 1999; Baumann et al., 2001). Sigel and colleagues have extended the use of double subunit and triple subunit concatemers to study the relative orientation and functional contribution of subunits within the pentamer (Baumann et al., 2002; Minier and Sigel, 2004). They propose the subunit arrangement to be γβαβα, counterclockwise when viewed from the synapse. The stoichiometry of receptors composed of only α and β subunits has also been assessed. The original tandem construct experiments of Im et al. (1995) suggested incorporation of 3 α6 subunits and 2 β2 subunits in pentameric α6β2 receptors. This stoichiometry for pentameric receptors composed of only α and β subunits is supported by immunoprecipitation studies (Kellenberger et al., 1996). However, results from others suggest the presence of 3 β subunits and 2 α subunits in αβ GABAARs (Tretter et al., 1997; Baumann et al., 2001; Horenstein et al., 2001). The different deduced stoichiometries may be due to the presence of distinct α subunit isoforms studied, or methodological differences.

2.3. Ion selectivity and channel conductance TM2 and the TM1-TM2 linker have been a major focus for assessing determinants of ion selectivity and channel conductance for all members of the Cys-loop LGIC superfamily. A number of seminal experiments and concepts are presented here. For a more extensive review of determinants of ion selectivity in this class of receptors, the reader is referred to a recent review by Jensen et al. (2005). Early studies by Galzi et al. (1992) on the nAChR α7 identified determinants of charge selectivity. By systematically mutating TM2 α7 subunit residues to those found in the GABAA α1 subunit, they identified three substitutions that collectively were sufficient to convert the channel from cation-selective to anion-selective. As illustrated in Fig. 1, these were: addition of Cation channels mnACh α7 m5-HT 3A

-5’ -2’ -1’ 0’ 6’ 9’ 13’ 19’ D S G E - K M M L S I S*V L*L*S L T V*F L L*V I V N S G E - R V S*F K I T*L*L L*G Y S V*F L*I*I*V S

Anion channels rGABA α1 rGABA β2 rGABA γ2 hGly α1

E D D D

S A A A

V S V A

P A P P

A A A A

R R R R

T V T V

T*F A L S L G L

G G G G

V I I I

T*T*V T T V T T V T T V

L*T*M L T M L T M L T M

T T T T

T*L T I T L T Q

S N S S

I*S*A T H L T I A S G S

R R R R

Figure 1. Alignment of the M1−M2 linkers and the M2 domains of selected subunits of the cl-LGICs. Each residue is labeled according to Miller (1989), where 0’ is the residue nearest the cystoplasmic region of the membrane. The residues involved in the determination of ion selectivity are shown in grey. Residues demonstrated to line the pore of the channel are denoted with an asterisk (*) (Akabas et al. 1994; Chini et al. 1994; Xu and Akabas 1996; Reeves et al. 2001). The GABAA β2 (Ymer et al. 1989) and γ2 (Shivers et al. 1989) subunits are included for discussion. The human glycine α (Bormann, Rundstrom et al. 1993) is shown for comparison. Further, residues that are bold have been shown to influence PTX sensitivity (Pribilla et al. 1992; Lynch et al. 1995; Lynch et al. 1997; Buhr et al. 2001; Das and Dillon 2005). (r, rat; h, human; m, mouse)

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a proline residue at the -2’ position (-2’P), mutation of glutamate to alanine at the -1’ position (E-1’A), and mutation of valine to threonine at the 13’ position (V13’T). Mutation of either individually did not confer anion-selectivity. The same mutations, when introduced into the cation-selective 5-HT3AR, also convert the channel to anionselective (Gunthorpe and Lummis, 2001). Keramidas et al. (2000) performed the opposite mutations in the anion-selective Gly α1 homomeric receptor (-2’P∆, A-1’E, and T13’V) and obtained results consistent with those of Galzi et al. (1992) and Gunthorpe and Lummis (2001); i.e., the mutations rendered the GlyRs cation-selective. Subsequent experiments have demonstrated the role of the -2’ and -1’ residues in charge selectivity of homomeric GABACRs (Wotring et al., 2003). Studies discussed to this point utilized homomeric receptors, which do not permit assessment of the relative contribution of each subunit to ion selectivity. Recently, Jensen et al. (2002) assessed whether the subunits in the α2β3γ2 GABAAR differentially contribute to charge selectivity. They introduced four or five TM1-TM2 linker residues (DSG-EK or SG-EK, with the dash representing deletion of the -2’ proline present in all anion-selective subunits, see Figure 1) present in the nAChR α7 subunit into either α2, β3 or γ2 subunits of the GABAAR, and expressed with wild-type of the companion subunits. Intoduction of this sequence into the β3 subunit was capable of conferring cation-selectivity, while its introduction into α2 or γ2 subunits did not alter selectivity. This demonstrates a subunit-dependent effect with regard to ion selectivity in GABAARs, and indicates two of the five subunits of the pentamer can control selectivity. Whether or not this subunit-selective influence will extend to all members of the Cysloop LGIC superfamily remains to be determined. Finally, Jensen et al. (2005) have recently used an engineered cation-selective GABAAR to further refine the essential characteristics of cation-selective versus anion-selective channels. They found that amino acid requirements at the TM1-TM2 linker to confer anion-selectivity were much less rigorous than those needed to confer cation-selectivity (Jensen et al., 2005). Their results led them to conclude that Cys-loop LGICs are by default anion-selective. In summary, results from numerous studies are consistent with the key role of the TM1TM2 linker in determining charge selectivity of members of the Cys-loop LGIC superfamily. Amino acid residues at the more extracellular aspect of the pore may also influence selectivity, but requirements at those positions appear less stringent than seen at the TM1-TM2 linker (Jensen et al., 2005).

2.4. The GABA binding site Structural domains involved in GABA binding are of obvious importance for understanding the channel gating process. Residues involved in GABA binding have been intensively studied in recent years. Because of a lack of a crystal structure for the GABAAR, researchers have had to rely on a number of indirect approaches to study GABA binding. These studies resulted in the discovery of several amino acid residues that were presumed to be critical for GABA binding, and an overall structural model of the ligand binding pocket. The discovery and solving of the structure of the AChBP by Sixma and colleagues (Brejc et al., 2001; 2002) was a significant breakthrough in the study of structure and function of LGICs. The AChBP is a 210 amino acid protein that forms a pentameric structure with binding domains for acetylcholine (ACh). Because of

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its high sequence similarity to all LGICs (15%-28% identity), the crystal structure of AChBP has become a high resolution model for the extracellular domain of nAChRs as well as other pentameric LGICs. This homology models provide a valuable template to analyze receptor-ligand interactions. Figure 2 shows sequence alignments of the ligand-binding domain of selected GABAAR and Torpedo nAChR subunits and the AChBP. Residues that are highly conserved within the superfamily are likely to be structurally equivalent between AChBP and other Cys-loop LGICs, while variable residues may reflect functional specialization and structural diversity. The ligand binding site of AChBP is characterized by the presence of aromatic and hydrophobic residues that are contributed by two neighboring subunits and a disulphide bond between two adjacent cysteine residues. The AChBP binding site pocket sits at the interface between two subunits. The principal subunit provides residues from loops A, B, and C, whereas residues within loops D, E, and F come from the complementary subunit mostly within β-strands. The structure of AChBP model provides a useful theoretical template to locate and identify GABA binding sites. The most common experimental approach to identify putative residues involved in GABA binding has been site-directed mutagenesis combined with functional and/or radioligand binding assays. A marked shift in the affinity for GABA and its competitive antagonists (bicuculline and gabazine) by mutation of specific extracellular residue provides first-line evidence for involvement of

Figure 2. Amino acid sequence alignment of the ACh-binding protein (AChBP) with the ligandbinding domain of the human GABAA receptor (G-A) β2, α1 and γ2 subunits, and Torpedo nicotinic acetylcholine (nACh) receptor α1 and γ subunits. Conserved cross-linked cysteines are yellow, while other fully conserved residues are in dark blue. Secondary structural elements of AChBP are denoted (α helix is pale blue; β strands are green). The approximate locations of the six domains involved in agonist binding (loops A-F) are also noted. Colored residues within those loops shown to be involved in binding are highlighted (From Cromer et al. (2002), with permission.)

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a particular residue in binding. In recent years, the SCAM has also been widely used to identify the residues that form the surface of the binding site. This approach has yielded new structural information regarding the orientation of ligand bound within the site. A homology model of the GABAARs based on AChBP revealed the presence of two functional GABA-binding sites at the β-α subunit interface by six loops A through F (Fig. 3A). There is evidence to suggest these two binding sites may not be functionally identical (Baumann et al., 2003). Loop A, B and C form the principal site and D, E and F form complementary site. Table 1 summarizes the residues that are to date identified to be involved in GABA binding according to the loop nomenclature system. Most experiments were conducted with recombinant α1β2γ2 combination. Similar to ACh binding sites, the binding residues experimentally identified to date also consists of aromatic (α1F64, β2Y62, β2Y97, β2Y157, β2Y205), hydroxylated (α1S68, β2T160, β2T202, β2S204, β2S209) and charged residues (α1R120, α1D183, α1R66, β2R207) (Table 1). These residues possess structural equivalents in modeling template AChBP in terms of position and secondary structure. By using SCAM method combined with homology modeling, it has been proposed that residues Y157 (loop B) and S204, Y205, R207 and S209 (loop C) at β2 subunit are critical for both agonist and antagonist binding (Amin and Weiss 1993; Wagner and Czajkowski, 2001). Loop A (Y97 and L99 region) at the β2 subunit and loop D (α1 F64-S68) form a β-strand. The residues from loops A, B, and C form one face of the binding pocket. The complementary face of the binding pocket is formed by residues from loops D and F (Fig. 2A). Figure 2B illustrates a molecular model for GABA binding sites. More detailed work suggests that residues in the binding pocket have different roles in ligand-binding. In the radioligand binding assay, the GABAARs show both high and low affinity binding sites for agonists. Low

Figure 3. The GABA binding site. (A) Cartoon representation of the GABA binding site, using the six-loop agonist binding site model (loop A-F). (B) The GABA-binding pocket of β/α interface viewed from the side. A molecule of GABA is shown in the Corey–Pauling–Koltun format, whereas residues involved in agonist binding are shown as ball-and-stick models. Fig. is produced, with permission, from Kash et al. ( 2004).

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Table 1. Residues identified to participate in GABA binding.

affinity sites are implicated to channel activation. Newell et al. (2000) found Y62 of the β2 subunit constitutes a high affinity binding site in loop D. Mutation of Y64 to F or S lost high affinity KD of [3H]muscimol labeling without apparently affecting [3H]flunitrazepam ([3H]FNZ) binding activity or GABA EC50 value. This high affinity binding site plays a role in stabilizing receptor desensitization (Newell et al., 2000). Furthermore, Wagner et al. (2004) show that β2 R207 at loop C stabilizes GABA in the binding pocket and may directly contact GABA molecule. Mutation of β2 L99 or E155 results in a spontaneous opening, suggesting these residues may be associated with coupling agonist binding to channel gating (Boileau et al., 2002; Newell et al., 2004).

2.5. Transduction of the gating process The obvious purpose of ligand binding is to initiate a conformational change that ultimately results in opening of the channel. Although the complete transduction process has not been elucidated, recent studies have uncovered components of this process. A short extracellular loop between TM2 and TM3 (so-called M2-3 linker) was proposed to link ligand-binding to channel gating in Cys-loop LGICs. Inherited mutations in this region for LGIC impair channel activation without altering ligand-binding (Kusama et al., 1994; Lynch et al., 1995; Rajendra et al., 1995; Campos-Caro et al., 1996; Baulac et al., 2001). In the crystal AChBP structure, loops 2 and 7 are positioned to directly interact with membrane. This lead to the hypothesis that these loops may interact with transmembrane domains to activate the channel (Brejc et al., 2001). Miyazawa et al. (2003) proposed a “pin-into-socket” mechanism for coupling binding to gating in the nAChR, based on the 4 Å structure. As illustrated in Figure 4Aa, in their hypothesis, binding of agonist causes a 15° clockwise rotation of the inner β-sheets of the extracellular binding region and passes through the cys loop (loop 7). This presumably brings a valine residue (V44) of the α subunit in loop 2 into contact with a hydrophobic pocket in the M2-3 linker. This initiates movement of the M2-3 linker, which is transmitted to the TM2 region and leads to opening of the channel gate (Miyazawa et al., 2003). Thus, in this model, α1V44 represents the sole physical link between the extracellular and transmembrane domains.

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Figure 4. Proposed model of the coupling mechanism from the binding site to gating for nACh (A) and GABAA receptors (B). (Aa) One subunit structure of nACh receptor is shown. The extracellular domain (top) couples to the transmembrane domain of the receptor through interaction of key functional groups, particularly loop 2 (L2) and loop 7 (L7). See text for description. (B) Proposed binding/gating coupling mechanism in the GABAA receptor α1 subunit. Ba shows a side view of the GABAA receptor α1 subunit, with key domains presented in different colors. A close-up view of the potential contacts between the M2-M3 segmanet (in magenta) and the transmembrane region (in red ribbons) is illustrated in Bb. See text for additional description. Panel A is produced, with permission, from Lester et al., 2004; Panel B is reproduced, with permission, from Kash et al., 2003.

A somewhat different mechanism has been proposed for transduction of the gating process in the GABAAR. Harrison and his colleagues (Kash et al., 2004) examined whether this pin-into-socket mechanism proposed for nAChRs also contributes to activation of GABAARs and GlyRs. They mutated residues corresponding to nAChR α1V44 in the GABAAR (α1H56 and β2V53) and GlyR (α1T54). The majority of mutations made at this position had little or no effect on receptor function. Their data do not support a simple pin-into-socket mechanism mediated by a single residue for activation of GABAARs and GlyRs (Kash et al., 2004). Instead, accumulating studies have shown that the M2-3 linker and the pre-TM1 region serve as mechanical receivers for the conformational changes initiated by occupation of the agonist-binding site (Lynch et al., 1997; Absalom et al., 2003; Kash et al., 2003). In the GABAAR α1 subunit, loops 2 and 7 contain negatively charged residues (D57, D279) and the M2-3 linker has a positively charged lysine (K279, Fig. 4Bb), which previously has been implicated in channel gating (Sigel et al., 1999). Kash et al. (2003) showed that substitution of K for D at α1D57 in loop 2 or α1D149 in loop 7 disrupted channel gating. Gating could be restored in the mutant receptors by substituting the positively charged K279 of the M2-3 linker with the negatively charged

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residue D (which produced a charge reversal double mutant). Using cysteine crosslinking, they were further able to demonstrate that α1D149 and α1K279 become closer with channel gating (Fig. 4Bb). These experiments suggest that in wild-type receptors, an electrostatic interaction between K279 of the α1 subunit M2-3 linker and negatively charged residues of extracellular loops 2 and 7 couple ligand binding to channel gating in the GABAAR. Interestingly, although an electrostatic mechanism exists for coupling binding to gating in GABAAR β2 subunits, residues distinct from those identified in the α1 subunit play the key role. K215 of the pre-TM1 region, not lysine residues of the M2-3 linker, interacts with the aspartic acid (D146 in the β2 subunit) of loop 7 (Kash et al., 2004). Thus, transduction of ligand binding in the extracellular domain to the initiation of gating in GABAARs can be briefly outlined as follows. The binding of agonist at the GABA binding pocket causes conformational changes which distribute the movements into the transmembrane domains. Loops 2 and 7 from α and β subunits interact with residues in the M2-3 linker and the pre-TM1 domain. These interactions initiate the movements that result in opening of the channel. These experiments have provided insight into the mechanism by which binding of ligand at a distant extracellular site couples to the transmembrane domain of the protein. The actual opening of the TM2 channel gate, however, occurs considerably deeper in the transmembrane domain (Fig. 1). Mutations in TM2 have demonstrated several residues are important in channel gating. Of particular importance appears to be the TM2 9’ position. It was demonstrated early on that mutating the 9’ leucine to threonine in neuronal α7 nAChRs reduced the rate of desensitization (Revah et al., 1991). Tierney et al. (1996) introduced an L9’T mutation into the β1 subunit of α1β1 GABAARs. These receptors were no longer gated by GABA, but were instead spontaneously open; GABA binding and channel gating were thus uncoupled. Similarly, mutation of the L9’ to S in any subunit of α1β2γ2 GABAARs resulted in left-shifted GABA concentration-response profiles (Chang and Weiss, 1999). Several other studies have confirmed a key role of the TM2 9’ position in GABAAR channel gating (Chang and Weiss, 1998; Dalziel et al., 2000; Ueno et al., 2000; Bianchi and Macdonald, 2001). A general thought is that the 9’ leucines interact with one another through hydrophobic forces in the closed stated, and this force is overcome with the conformational events associated with channel gating. Substitution of the 9’ leucines with smaller residues, or those less attracted to one another, leads to a reduced tendency to hold the channel in the closed state, and thus enhanced gating capabilities. A number of other TM2 residues also significantly impact channel gating, including the 15’ and 6’ positions. As with the 9’ position, mutations of the 15’ position can also elicit shifts in agonist sensitivity and desensitization kinetics (Koltchine et al., 1999; Scheller and Forman, 2002). In studies of homomeric β1 and β3 GABAARs expressed in Xenopus oocytes, the 15’ residue was also shown to be important in spontaneous activity and anesthetic modulation (Cestari et al., 2000). Subsequent work has shown that spontaneous activity induced by mutations at the 15’ position correlates with molecular size of the amino acid substitution (Miko et al., 2004). The TM2 6’ position also contributes to channel gating. Horenstein et al. (2001) observed that the T6’C mutations underwent covalent cross-linking between mutant α1 and β1 subunits. Lynch and colleagues (Shan et al., 2002) determined that the T6’C

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mutation in α1β1 GABAARs also conferred rapid desensitization kinetics in α1(T6’C)β1(T6’C) receptors. Work in other Cys-loop receptors supports the findings that the TM2 6’ position influences gating kinetics. Receptors with the autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) mutation (S6’F), are characteristically quicker to desensitize than the wild type receptor (Weiland et al., 1996; Bertrand et al., 1998). Additional residues that line the channel pore have been identified to influence channel kinetics, although studies to determine the effect these residues have on the GABAAR has not been conducted (Milone et al., 1997; Gunthorpe et al., 2000).

3. Allosteric modulation of the GABAA receptor

The activity of the GABAAR is influenced by numerous agents which are of importance from a clinical and/or basic science perspective. Knowledge of particular domains important in the binding and functional effects of many of these ligands to the GABAAR has grown considerably in recent years. Our current understanding of key structural regions for the interaction of several of these ligands with the GABAAR follows.

3.1. Benzodiazepines Benzodiazepines (BZDs) act as anxiolytics, anticonvulsants, sedative/hypnotics and muscle relaxants, and do so by potentiating the actions of GABA. BZDs bind to the GABAAR and allosterically modulate GABA-gated currents. Besides BZDs, many classes of structurally diverse compounds can also bind to the BZD site. Depending on the ligands and the subunit composition of the GABAAR, BZD ligands can potentiate (positive modulators like diazepam, flunitrazepam and chlordiazepoxide), inhibit (negative or inverse agonists like Ro15-4513 or some β-carbolines) or have no effect (BZD antagonists or null modulators like flumazenil) on GABA currents. BZD positive modulators shift the GABA concentration response curve to the left but do not increase maximal GABA responses. In contrast, BZD negative modulators increase GABA EC50. BZD antagonists competitively block the effect of both positive and negative modulators but lack any intrinsic modulatory effects on the receptor ion channel complex. Because BZD ligands are among the most widely used drugs, understanding the structure of BZD binding site is of great therapeutic relevance. As described above, GABA binding sites lie at the α-β subunit interfaces. Homologous residues at the α and γ interfaces form BZD binding sites in GABAARs (Sigel and Buhr, 1997). Using chimeric and site-directed mutagenesis, SCAM and photoaffinity labeling methods, H101, Y159, Y161, T162, G200, T206 and Y209 on the α1 subunit and Y58, F77, A79, T81 and M130 on the γ2 subunit have been shown to contribute to the binding pocket of BDZs (Pritchett and Seeburg, 1991; Wieland et al., 1992; Duncalfe and Dunn, 1996; Amin et al., 1997; Buhr et al., 1997; Buhr and Sigel, 1997; Wingrove et al., 1997; Schaerer et al., 1998; Sigel et al., 1998; Kucken et al., 2000; Teissere and Czajkowski, 2001). Many of these residues are in equivalent or adjacent positions to those that form the GABA binding site on the α1 and β2 subunits. For example, γ2 F77, α1Y159, α1T206 and α1Y209 in BZD-binding pocket are directly homologous to α1 F64, β2Y159, β2T202 and β2Y205 in the GABA-binding pocket, respectively (for review, see (Sigel and Buhr, 1997; Cromer et al., 2002). A histidine

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residue (H101 in the α1 subunit) in α1, α2, α3 and α6 confers binding ability for classic BZDs whereas an arginine at homologous position of α4 and α6 is mainly responsible for BZD insensitivity of α2/6β2γ2 receptors (Wieland et al., 1992; Wieland and Luddens, 1994). Mutational studies show that the region of γ2 F77-T81 in the γ2 subunit is important for BZD ligand discrimination (Buhr and Sigel, 1997; Kucken et al., 2000; Cope et al., 2004). The γ2 F77I point mutation abolishes sensitivity to zolpidem but not to other BZD agonists (Buhr et al., 1997; Cope et al., 2004). As for other allosteric GABAAR modulators, exactly how the binding of BZDs is coupled to changes in GABA binding and/or GABA activation of the channel remains unknown. According to allosteric theory, there could be allosteric cross-talk between the BZD binding site and the agonist (GABA) binding site. Teissere and Czajkowski (2001) used SCAM to demonstrate the region γ2 Y72-Y83 in the BZD binding pocket undergoes an allosteric structural arrangement during GABA binding and channel gating. Furthermore, modification of the BZD binding site by sulfhydryl-specific reagents allosterically shifts the GABA sensitivity (Teissere and Czajkowski, 2001). A more definitive understanding of the influence of BZDs on GABA binding and gating awaits additional study.

3.2. Barbiturates The barbiturates, one of the oldest classes of anesthetics, were introduced for intravenous anesthesia in the early 1900s. It was known early on that pentobarbital and thiopental could mediate central nervous system depression (Goldstein and Aronow, 1960). Despite their extensive history, the biological target of anesthetics was not known for decades. Introduced in the early 1900s, the lipid theory of anesthetic action held sway for decades. It was only in more recent times that a small set of targets within the nervous system were shown to be specific targets of various anesthetic agents (Franks and Lieb, 1994). In the case of barbiturates, Evans demonstrated that GABA-activated current of isolated rat superior cervical ganglia was modulated by pentobarbital (Evans, 1979). Further work in frog sensory neurons demonstrated the ability of pentobarbital to potentiate GABA-gated current (Akaike et al., 1985). Study and Barker (1981) used fluctuation analysis to determine that pentobarbital increases the mean open time of GABAARs in the presence of GABA. As with other modulatory agents, the discovery of different subunits of the GABAAR has permitted assessment of subunit-dependence of barbiturate modulation of GABAARs. Thompson et al. (1996) used varied combinations of α and β subunits, expressed with the γ2 subunit, to characterize the action of pentobarbital. Their results led them to conclude that the degree of potentiation depended on the α subunit present (Thompson et al., 1996). These studies also provided evidence to suggest that the α and β subunits of the GABAAR contribute to a pentobarbital site of interaction. In αβδ GABAARs, which are largely extrasynaptic, pentobarbital introduces a third open state and enhances the effects of saturating concentrations of GABA, which the authors attributed to the low efficacy of GABA in these receptors (Feng et al., 2004). Barbiturates also have the ability to directly activate the GABAAR (Robertson, 1989; Franks and Lieb, 1994), and this effect also shows subunit-dependence. Receptors containing the α6 subunit display the highest potency, while the β subunit has the

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greatest influence on barbiturate efficacy (Thompson et al., 1996). Rho et al. (1996) showed that pentobarbital-activated currents were inhibited by picrotoxin and the deactivation kinetics of these receptors was similar to GABA-gated currents, suggesting ultimately similar gating mechanisms. A rebound current is observed at high concentrations of barbiturate, and likely reflects unbinding from a low affinity inhibitory site (Akk and Steinbach, 2000). Despite considerable study, a binding site for barbiturates on GABAARs has not been identified. Arias et al. (2001) reported the presence of multiple barbiturate sites in nAChRs from Torpedo californica. Thus, multiple barbiturate sites in the GABAARs are feasible. Chimeric GlyRs/GABAARs have been used to assess possible domains involved in barbiturate sensitivity (Koltchine et al., 1996). The authors identified two regions, a C-terminal fragment including the cytoplasmic loop and TM4, and a smaller N-terminal fragment, that were not responsible for either modulatory or direct gating effects of the barbiturate methohexital. Although a binding site has not been defined, several residues have been identified that influence barbiturate sensitivity. In a study of α2β1 GABAARs, mutation of a TM3 residue in either subunit (A291 in α2, M286 in β1) significantly decreased the enhancing actions of the barbiturate methohexital (Krasowski et al., 1998). In α6β3γ2 receptors expressed in Xenopus oocytes, mutation of the β subunit TM2 15’ asparagine reduced the efficacy of pentobarbital (Pistis et al., 1999). In addition, mutation to phenylalanine of a glycine residue at the beginning of the TM1 domain (G219) of the GABAAR β2 subunit eliminates pentobarbital enhancement of α1β2γ2 receptors (Carlson et al., 2000). The disparate locations of these residues suggest they may not be part of a binding site per se, but are involved in transduction of the actions of barbiturates.

3.3. General anesthetics In addition to the anesthetic steroids and the barbiturates, several general anesthetics of diverse structure such as volatile anesthetics (e.g., iosflurane, enflurane, halothane) and intravenous anesthetics (e.g., etomidate, chlormethiazole and propofol), are among the most widely used which primarily interact with GABAARs. Like barbiturates and neurosteroids, etomidate and propofol both potentiate the effects of GABA and directly open the GABAAR at high concentrations. Several lines of evidence support the idea that the a binding pocket, partly defined by the residues at the extracellular end of TM1, TM2 and TM3 helices, may exist for general anesthetics (Belelli et al., 1997; Mihic et al., 1997; Krasowski et al., 1998; Carlson et al., 2000; Siegwart et al., 2002). In light of the observation that most anesthetics do not enhance the ρ subunit of GABAARs, studies using chimerical constructs and site-directed mutagenesis have identified at least three domains at this region critical to modulatory effect of general anesthetics (Fig. 5) (Belelli et al., 1997; Sanna et al., 1997; Carlson et al., 2000; Chang et al., 2003). These anesthetic binding domains consist of two pairs of aligned positions α1/2 S270 and β3 N265 in TM2 (15’), α1/2 A291 and β2/3 M286 in TM3, and β2 G219 in TM1 (Fig. 5). Replacing these residues in with the residues from ρ subunits (see Fig. 5) leads to a loss of enhancement by anesthetics (Belelli et al., 1997; Sanna et al., 1997). Moreover, the anesthetic direct activation of GABAAR may not be mediated through the same sites of the receptor

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subunits as mutation β2 (M286W) abolished modulatory action of propofol, etomidate and enflurane but it did not affect direct activation (Krasowski et al., 2001; Siegwart et al., 2003). The β3 N265 seems to be involved in both modulatory and direct effects. Mutation of this residue to methionine (N265M) largely abolished the direct and modulatory actions of etomidate and propofol at recombinant GABAARs (Belelli et al.,1997; Pistis et al., 1999; Siegwart et al., 2002). In contrast, the modulatory effects of the neurosteroidal anesthetic alphaxalone are preserved (Siegwart et al., 2002). Furthermore, a point mutation of β3 (N256M) “knocked-in” to mice totally eliminated the modulatory and direct effects of the intravenous anesthetics etomidate and propofol and slightly reduced modulatory actions of volatile anesthetic enflurane (Jurd et al., 2003; Rudolph and Mohler, 2004). Another domain to participate in anesthetic action is the TM1 region. When a TM1 glycine residue (G219 in β2 subunit), which is conserved across GABAAR subunits, is replaced with F, the amino acid found in ρ (Fig. 5), it eliminates modulatory effects of not only propofol and etomidate but also barbiturates and neuroactive steroid (Carlson et al., 2000; Chang et al., 2003). Using the AChBP model, this TM1 glycine residue is predicted to be positioned several Å away from two domains in TM2 and TM3, potentially making interactions with pore lining TM2 during channel gating and modulation by allosteric ligands (Chang et al., 2003; Olsen et al., 2004). The mechanism for anesthetic action is not clear. Homomeric α1 GlyRs display similar sensitive to the anesthetics through the action sites formed by the homologous residues on GABAA subunits (Fig. 5) (Wick et al., 1998; Ueno et al., 2000). SCAM studies on the TM1, TM2 and TM3 residues of GlyR α1 subunit that are involved in alcohols and anesthetics action suggest that drugs occupying in these region produces conformational changes during channel gating, which may stabilize the open state of the channel (Lobo et al., 2004).

Figure 5. An alignment of human glycine α1, GABAA α1, α2, β1-3 and ρ1 amino-acid sequences between TM1 and TM3. *, 10 residues that are identical in glycine and GABAA subunits but different in ρ1. #, the residues which differ between GABAA α/β and ρ1. The residues (bold, underlined) have been identified to be critical for anesthetic action in GABAA receptors (see text for details).

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3.4. Picrotoxin Picrotoxin (PTX), a proconvulsant plant alkaloid known for decades to inhibit GABAARs (Takeuchi and Takeuchi, 1969), has generally been considered a GABAAR antagonist. It was subsequently demonstrated that PTX inhibits other anion-selective LGICs, including GlyRs and GABACRs (Pribilla et al., 1992; Wang et al., 1995) and glutamate-gated Cl- channels (Etter et al., 1999). At somewhat higher concentrations (roughly 10-fold), PTX also inhibits cation-selective LGICs (Das et al., 2003; Erkkila et al., 2004). The mechanism of PTX-mediated antagonism is complex. Binding studies have shown that PTX does not compete for GABA or Gly binding sites (Ramanjaneyulu and Ticku, 1984; Lynch et al., 1995). The fact that PTX -induced blockade of the GABAAR is use-dependent led to the conclusion that it acts within the channel pore to directly block the current flow (Inoue and Akaike, 1988). Indeed, the association rate for PTX is greatly facilitated by channel opening (Dillon et al., 1995). Moreover, dissociation of PTX from its binding site is also facilitated by channel activation (Newland and CullCandy 1992; Dillon et al., 1995). However, PTX can gain at least minimal access to its binding domain in the absence of channel opening (Dillon et al., 1995). In addition, Lynch et al. (1995) found little use-dependent block by PTX of recombinant human α1 GlyRs. The pharmacological nature of PTX blockade shows both competitive and noncompetitive components (Smart and Constanti, 1986; Krishek et al., 1996; Yoon et al., 1998; Das et al., 2003; Qian et al., 2005). Because PTX does not compete at the ligand binding site (Ramanjaneyulu and Ticku, 1984; Lynch et al., 1995), the competitive component of block is not true competition in the classical sense, but more likely an allosteric antagonism that appears competitive functionally (Bertrand et al., 1992; Lynch et al., 1995). The presence of both competitive and non-competitive aspects of PTXmediated blockade underscores the complex interactions of this drug with its binding domain(s) in ligand-gated ion channels. Although a site of PTX binding has not been established definitively, there is considerable evidence indicating it interacts at the second transmembrane domain of these LGICs. Pribilla et al. (1992) demonstrated that the relative insensitivity of αβ GlyRs to PTX is conferred by a region of the β subunit TM2 domain. Shortly thereafter, Reddy et al. (1993) demonstrated that insertion into a lipid bilayer of a 23 amino acid peptide with a sequence equivalent to TM2 of the GlyR α subunit produced spontaneously open channels that could be blocked by PTX. Around the same time, ffrench-Constant and colleagues (ffrench-Constant et al., 1991; ffrench-Constant, 1993) demonstrated that resistance to the insecticide dieldrin and PTX in the Drosophila strain Rdl (resistant to dieldrin) is due to a natural mutation (A→S or A→G) at the TM2 2’ position. The residues which influence PTX sensitivity are summarized in Fig. 1. The ability of PTX to prevent sulfhydryl reaction of a cysteine residue engineered into the 2’ position of the GABAAR also supports the hypothesis that PTX binds in this region of the channel (Xu et al., 1995). Finally, the 2’ position also influences picrotoxin sensitivity in GABACRs (Enz and Bormann, 1995; Wang et al., 1995) and glutamategated Cl- channels of nematodes (Etter et al., 1999). Whereas some role of the TM2 2’ position in PTX -mediated inhibition is likely, further research has presented compelling evidence that the TM2 6’ position plays the

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key role in the actions of PTX. Based on the results of Pribilla et al. (1992), Gurley et al. (1995) sought to define TM2 residue(s) responsible for PTX blockade of GABAARs. They demonstrated that substitution of a phenylalanine residue (wild type is threonine) at the 6’ position in TM2 of the β2 subunit of α1β2γ2 GABAARs produces resistance to PTX. Zhang et al. (1995) also demonstrated a role of the 6’ position in PTX sensitivity of GABACRs. More recently, Shan et al. (2001) assessed the potential involvement of both 2’ and 6’ TM2 residues in PTX inhibition of homomeric and heteromeric GlyRs. Their results demonstrated the presence of a ring of threonine residues at the 6’ position is important for high affinity PTX inhibition. Our recent results (Das and Dillon, 2005) also demonstrate a crucial role for the TM2 6’ position in PTX -mediated inhibition of 5HT3Rs. The molecular basis underlying the importance of the TM2 6’ residue has not been defined. Zhorov et al. (2000) proposed that the 6’ position threonine acts as a hydrogen bond donor to stabilize the hydrophilic end of the PTX molecule in the channel. In addition to a presumed site of PTX in the ion channel, there is considerable evidence that PTX may also interact with a second site. Results from studies of Yoon et al. (1993) led them to conclude that PTX acts at a use-dependent site and a useindependent site in GABAARs. They suggested the use-dependent site may exist outside of the channel but in a location such that it is modified by ligand binding and channel gating, while the use-independent site may be distant from the agonist recognition site. A number of studies have provided more specific evidence of a potential second site for picrotoxin. Lynch et al. (1995) found that mutations in the human glycine α1 (R271L or R271N, the 19’ position in TM2) subunit that cause startle disease convert PTX from a competitive antagonist into an allosteric agonist. Their results led them to conclude that PTX not only has two distinct functions, but interacts at two distinct binding sites. Perret et al. (1999) studied the ability of chemically reactive probes derived from noncompetitive blockers of GABAARs to interact with TM2 residues of the α1 subunit. The sulfhydryl-reactive probes, derived from molecules that likely interact at the PTX site, were exposed to GABAARs carrying cysteine substitutions in several TM2 residues. Irreversible cross-linking was obtained with cysteines at both the TM2 2’ and TM2 6’ positions, suggesting the non-competitive blockers interact at this region. They also found that PTX and the putative PTX-site ligand t-butylbicyclophosphorothionate (TBPS) could protect the 2’ position from modification by the reactive probes. These results are consistent with those of Xu et al. (1995) who also found PTX protects the 2’ position from sulfhydryl modification. Additionally, however, Perret et al. (1999) found that the reactive non-competitive antagonists could also react at the TM2 17’ position; this covalent modification could also be blocked by PTX. They thus concluded that PTX binds at two sites, one deep in the channel and one in the vicinity of the TM2 17’ position. Additionally, the demonstration that mutations of the TM2 15’ position in α1 GlyRs convert PTX -mediated inhibition from competitive, non-use-dependent to noncompetitive and use-dependent further argue for the interaction of PTX at a second site (Dibas, Gonzales et al. 2002). It is also of note that PTX and other PTX-site ligands have been shown to have stimulatory actions in a number of cases (Newland and Cull-Candy, 1992; Nagata et al., 1994; Nagata and Narahashi, 1994; Lynch et al., 1995; Bell-Horner et al., 2000). PTXmediated stimulation of ligand-gated channel activity can be interpreted mechanistically

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in several ways, only one of which would necessarily incorporate two distinct binding sites. Collectively, however, the data favor the possibility that PTX interacts with two separate domains in LGICs.

4. Endogenous physiologic modulators of the GABAA receptor

In addition to possessing modulatory sites for several exogenously derived agents, activity of the GABAAR is also influenced by many endogenous stimuli. The ability of several of these to modulate GABAergic function is described here.

4.1. Zn2+ and other divalent cations It has been known for some time that GABAARs are negatively modulated by divalent cations such as Zn2+, Cu2+, Cd2+, Ni2+ Mn2+ and Co2+ whereas other metal cations such as Ca2+, Mg2+ or Ba2+ generally have little direct effect (Celentano et al., 1991). Of those metals that actively modulate GABAARs, Zn2+ and Cu2+ have attracted much attention because they are important trace metals that are differentially distributed throughout the mammalian CNS. Both Zn2+ and Cu2+ are stored in synaptic vesicles and released by electrical depolarization, reaching a high micromolar concentration at the synaptic cleft (Howell et al., 1984; Hartter and Barnea, 1988). They are believed to play physiological roles in regulating synaptic activity and neuronal excitability (Horning and Trombley, 2001). The actions of Cu2+ and in particular Zn2+ on GABAARs have been extensively studied and summarized by several recent review articles (Mehta and Ticku, 1999; Korpi et al., 2002; Smart et al., 2004). Here we focus on recent developments in the mechanism and structural determinants of these cations with respect of their modulation of GABAARs. In Zn2+/Cu2+ bound protein, Zn2+ or Cu2+ ions interact directly with two to four amino acids. Histidine, cysteine, aspartate or glutamate is commonly found to bind to these metals (Higak et al., 1992; Regan, 1993). A histidine residue that is conserved in all β subunits (H267) but not at other subunits was the first residue identified as a high affinity binding site for Zn2+ inhibition (Wooltorton et al., 1997; Horenstein and Akabas, 1998). Hosie et al. (2003) identified four additional extracellular residues, α1 E137, α1 H141, β3 E182 and β3 E270, that also participate in Zn2+ binding. They proposed that Zn2+ inhibits GABAARs at three discrete binding domains. One is near to the extracellular end of TM2 and requires β3 H267 and β3 E270. The other two exist on Nterminal sites at the α-β interface and each comprises α1 E137, β3 E137 and β3E182. The authors hypothesize that Zn2+ inhibits GABAARs by binding to these three different domains and stabilizing channel closure. Mutation of all three Zn2+ sites completely abolishes Zn2+-induced inhibition. In addition, as illustrated in Fig. 6, αβ receptors have three Zn2+ binding sites, which display a higher sensitivity to Zn2+. Incorporation of the γ2 subunit into the receptor results in loss of two of the extracellular Zn2+ sites, making the αβγ receptors much less sensitive to Zn2+. Indeed, replacing selected residues in the γ2 subunit with Zn2+ binding residues in the β3 subunit restored the 99% of the Zn2+ sensitivity that was lost in αβγ receptors, further supporting their hypothesis. The divalent cation Cu2+ has been also recognized as a potent inhibitor of GABAARs (Fisher and Macdonald, 1998; Kim and Macdonald, 2003). Inhibition of GABAARs by Zn2+ is partially relieved by the presence of Cu2+, suggesting Zn2+ and Cu2+ sites may

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Figure 6. Inhibition of α1β3 and α1β3γ2S GABAA receptors by Zn2+. Binding sites for GABA and benzodiazepines are also shown. In α1β3 receptors, three binding sites (two on the extracellular aspect of the receptor and one in the mouth of the channel) exist for Zn2+ (black circles). The presence of the γ2 subunit disrupts two of the three Zn2+ binding sites, thus conferring reduced sensitivity in α1β3γ2S receptors. Figure is produced, with permission, from Hosie et al. (2003).

interact or overlap (Sharonova et al., 2000; Zhu et al., 2002). Cu2+ inhibition seems to be α subtype dependent as α1β3γ2L receptors are more sensitive to Cu2+ than α6β3γ2 receptors (Fisher and Macdonald, 1998). An N-terminal motif containing V134, R135 and H141 in the α1 subunit confers higher Cu2+ sensitivity. α1 H141, which is not found at aligned position of other α subunits, is a major determinant in this motif. Furthermore, H267 on the β subunit, which contributes to one of the Zn2+ binding sites (see above) also, contributes to Cu2+ inhibition. Another histidine residue in the M2-3 linker, present only in α4/6 subunits (H273), influences Cu2+ and Zn2+ inhibition as well (Kim and Macdonald, 2003). Whether other Zn2+ binding sites and/or unidentified sites are also involved in Cu2+ inhibition is to be determined.

4.2. Protons pH in the brain changes with neural activity. In physiological conditions, a brain pH of 6.5 to 8.0 may exist (Kaila and Ransom, 1998). In certain pathological conditions, brain pH can even shift up to 1 unit (Siesjo et al., 1985; von Hanwehr et al., 1986). It has been well-known that H+ modulates neuronal excitability and this effect is partially mediated through pH modulation of GABAARs. The reports on H+ modulation of mammalian GABAARs vary from different preparations depending on the experimental conditions, animal species, brain regions or subunit compositions (Tang et al., 1990; Pasternack et al., 1992; Smart, 1992; Vyklicky et al., 1993; Krishek et al., 1996; Zhais et al., 1998; Huang and Dillon, 1999; Krishek and Smart, 2001; Wilkins et al., 2002; Li et al., 2003; Mozrzymas et al., 2003; Feng and Macdonald, 2004; Huang et al., 2004). The results from our laboratory and others have shown that acidic H+ inhibits GABA response recorded from recombinant receptors and several brain regions (Smart, 1992; Zhai et al., 1998; Huang and Dillon, 1999; Li et al., 2003; Huang et al., 2004). The

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down-regulation of GABAARs by acidosis is also consistent with the observation of depressant GABAergic function following ischemia insult (Green et al., 2000). Although the mechanism and sites for H+ -mediated inhibition of GABAARs remain to be fully elucidated, several lines of evidence support the hypothesis that pH modulation occurs in part by influencing GABA binding, either directly or allosterically. Our reports (Huang and Dillon 1999; Huang et al. 2004) and those of others (Zhai et al., 1998; Li et al., 2003) demonstrated that the H+ effect is competitive with respect to GABA and its competitive antagonist bicuculline. H+ did not affect GABAAR-mediated single-channel conductance (Huang and Dillon, 1999; Krishek and Smart, 2001). Changes in pH do not affect channel gating by pentobarbital or spontaneous chhannel opening in α1(L264T)β2γ2 receptors (Huang et al., 2004). [3H]GABA binding in mammalian brain membranes is decreased with a decrease in pH over the range 5.8 to 8.0 (Olsen et al., 1981). By systemically mutating the GABA-binding residues, we identified two extracellular residues, α1F64 and β2Y205, are involved in the inhibition of α1β2γ2 GABAARs by acidic pH. In addition, since H+ action site is found to share or overlap with Zn2+ sites in GlyRs (Chen et al., 2004), we also tested this hypothesis in the GABAARs by mutating Zn2+ -binding sites. Individual mutation of Zn2+ binding residues (i.e., α1 E138, β2 E182, β2 H267 and β2 E270) failed to influence pH sensitivity of GABAARs (Huang et al., 2004). Our data are contradictory to a study which shows that one Zn2+ binding residues, H267 in the TM2 of β2 subunit, confers the H+ modulation in α1β1/2 receptors (Wilkins et al., 2002). It appears that pH modulates GABAARs through multiple sites and even multiple mechanisms in the different receptor configurations.

4.3. Neurosteroids Neurosteroids are neurally active steroid molecules. They may be metabolites of reproductive or stress hormones, or they may be synthesized de novo in the CNS. A number of studies in the mid 1980’s demonstrated the GABAAR is a target of neurosteroids (Harrison and Simmonds, 1984; Majewska et al., 1986; Callachan et al., 1987), depending on the specific neurosteroid, the effect on GABAAR activity can be either stimulatory or inhibitory. At sufficiently high concentrations, several neurosteroids can also directly activate the GABAAR (Cottrell et al., 1987). For a number of reasons, including their presence endogenously and their relatively high affinity and efficacy, these compounds are of considerable interest as potential therapeutics. As with other allosteric modulators, the question of subunit dependence is of interest for the neurosteroids. A recent review by Lambert et al. (2003) nicely summarizes current knowledge of subunit-dependent steroid modulation of the GABAAR. Thus, only a brief synopsis is presented here. Lambert et al. (2003) report that the maximal stimulatory actions of the progesterone metabolite 5α-pregnan-3α-ol-20-one (5α3αTHPROG) are not significantly dependent on the α subunit isoform. They further note, however, that the 3-4 fold differences in EC50 values observed with different α subunit isoforms may have important physiological relevance. With regard to the β subunit, it appears to have relatively little influence on steroid modulation of GABAARs. However, direct activation of the receptor by the steroid anesthetic alphaxalone is dependent on the β subunit (Siegwart et al., 2002). The γ subunit is not required for neurosteroid’s actions on GABAARs, although it influences potency. Alphaxalone potentiates GABA-mediated

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current more efficaciously and with higher potency in receptors containing the γ3 subunit (Maitra and Reynolds, 1999). With regard to allopregnanolone, efficacy but not potency is affected by the γ subunit isoform (Maitra and Reynolds, 1999). Experiments in recent years have suggested that the δ subunit plays an important role in steroid modulation of the GABAAR. As noted above, δ-containing receptors are largely extrasynaptic and are known to be a major determinant of tonic background current that regulates neuronal excitability. Although an early report suggested the δ subunit depressed the effects of neurosteroids (Zhu et al., 1996), more recent work has suggested the opposite. For instance, δ subunit knockout mice are no longer sensitive to neuroactive steroids (Mihalek et al., 1999). In addition, Pillai et al. tested a wide variety of pregnane compounds on both γ and δ containing GABAARs; most of the compounds were more efficacious in the δ containing receptors (Pillai et al., 2004).

4.4. Protein phosphorylation Like many ion channels and receptors, the GABAAR is susceptible to regulation by phosphorylation. A number of protein kinases such as protein kinase C (PKC), protein kinase A (PKA), protein kinase G (PKG), tyrosine kinase and Ca2+/calmodin-dependent kianse II (CAMKII), have been reported to modulate GABAARs by direct phosphorylation of residues at the large intracellular loop between TM3 and TM4 (see reviews by Moss and Smart, 1996; Smart, 1997; Brandon et al., 2002; Kittler and Moss, 2003; Mody and Pearce, 2004). Recent studies have shown that, in addition to the modulation by direct phosphorylation, protein kinase-mediated modulation of surface receptor trafficking could also be an important mechanism for GABAAR plasticity. One example is PKC-mediated receptor endocytosis. Down-regulation of GABAAR function by PKC activation has been well-documented in the Xenopus oocyte expression system (Sigel and Baur, 1988; Kellenberger et al., 1992; Leidenheimer et al., 1992; Chapell et al., 1998; Filippova et al., 2000). This down-regulation by PKC may be mediated by direct phosphorylation and/or receptor endocytosis (Kellenberger et al., 1992; Chapell et al., 1998; Filippova et al., 2000). However, the results from studies with mammalian expression system (e.g., HEK293) or neuronal preparation are controversial. Activation of PKC has been shown to enhance (Lin et al., 1996), inhibit (Connolly et al., 1999; Cinar and Barnes, 2001) or have no effect (Ticku and Mehta 1990; Huang et al., 1999) on GABA-gated currents. One of the interpretations for these discrepancies is the experimental temperature. Whereas endocytosis is very limited at room temperature in mammalian cells, it proceeds efficiently in the Xenopus oocyte. Indeed, PKC-induced internalization of GABAARs has been observed at room temperature in the Xenopus oocyte (Chapell et al., 1998; Filippova et al., 2000). In contrast, the ability of PKC to inhibit GABAAR function (via endocytosis) is virtually lost when the recording temperature is lowered from 35°C to room temperature in the HEK293 expression system (Machu et al., 2005). The target and mechanism of PKC in the modulation of receptor endocytosis is not fully understood. PKC-mediated endocytosis is not dependent on direct receptor phosphorylation because mutation of the known PKC sites on the receptors (S409 in β1/3 and S410 in β2) does not alter PKC effect on endocytosis (Chapell et al., 1998; Connolly et al., 1999). PKC-mediated endocytosis is clathrin-mediated, and dependent on an AP2 adaptin dileucine-binding motif in intracellular loop of the β

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subunit (L343/L344 in β2) (Herring et al., 2003). Since GABAAR surface number is critically involved in the efficacy of the synaptic inhibition (i.e., the amplitude of IPSPs) and PKC is involved in many signaling pathways, modulation of receptor internalization by PKC may have physiological significance for intracellular cross talk between GABAARs and other receptors such as G-protein-coupled receptors (Brandon et al., 2002) and tyrosine kinase receptors mediated by brain-derived neurotrophic factor (BDNF) (Jovanovic et al., 2004).

5. Conclusion Work in recent years has greatly expanded our understanding of the overall structure of GABAAR, the most abundant inhibitory neurotransmitter receptor in the CNS. Knowledge of domains critical for channel gating, conductance and selectivity continues to expand. Moreover, efforts to understand the involvement of specific subunits in a number of physiologic and pathophysiologic processes are yielding important benefits. Considerable work remains to be done, however. For instance, although we know much about GABA binding and the mechanism by which the extracellular domain couples to the transmembrane domains, conformational changes occurring between those two events, as well as those that ultimately open the channel gate, remain unclear. Research over the next several years will likely solve these remaining questions of channel gating, and continue to refine our understanding of pharmacologic and physiologic modulation of this critical receptor.

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