Drug interactions at GABAA receptors

Progress in Neurobiology 67 (2002) 113–159

Drug interactions at GABAA receptors Esa R. Korpi a,∗ , Gerhard Gründer b , Hartmut Lüddens b a

Department of Pharmacology and Clinical Pharmacology, University of Turku, Itäinen Pitkäkatu 4B, FIN-20520 Turku, Finland b Department of Psychiatry, University of Mainz, D-55131 Mainz, Germany Received 2 January 2002; accepted 27 March 2002

Abstract Neurotransmitter receptor systems have been the focus of intensive pharmacological research for more than 20 years for basic and applied scientific reasons, but only recently has there been a better understanding of their key features. One of these systems includes the type A receptor for the ␥-aminobutyric acid (GABA), which forms an integral anion channel from a pentameric subunit assembly and mediates most of the fast inhibitory neurotransmission in the adult vertebrate central nervous system. Up to now, depending on the definition, 16–19 mammalian subunits have been cloned and localized on different genes. Their assembly into proteins in a poorly defined stoichiometry forms the basis of functional and pharmacological GABAA receptor diversity, i.e. the receptor subtypes. The latter has been well documented in autoradiographic studies using ligands that label some of the receptors’ various binding sites, corroborated by recombinant expression studies using the same tools. Significantly less heterogeneity has been found at the physiological level in native receptors, where the subunit combinations have been difficult to dissect. This review focuses on the characteristics, use and usefulness of various ligands and their binding sites to probe GABAA receptor properties and to gain insight into the biological function from fish to man and into evolutionary conserved GABAA receptor heterogeneity. We also summarize the properties of the novel mouse models created for the study of various brain functions and review the state-of-the-art imaging of brain GABAA receptors in various human neuropsychiatric conditions. The data indicate that the present ligands are only partly satisfactory tools and further ligands with subtype-selective properties are needed for imaging purposes and for confirming the behavioral and functional results of the studies presently carried out in gene-targeted mice with other species, including man. © 2002 Elsevier Science Ltd. All rights reserved.

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. GABAA receptor basics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Molecular biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Assembly and membrane targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Channel opening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: ␣6+/+ , ␣6 subunit wild-type mouse line; ␣6−/− , ␣6 subunit knockout mouse line; 5␣-DPH, 5␣-pregnan-3,20-dione; Allopregnanolone, 3␣-hydroxy-5␣-pregnan-20-one; ANT, alcohol non-tolerant rat line; AT, alcohol tolerant rat line; ␤-CCE, ethyl-␤-carboline-3-carboxylate; ␤-CCM, methyl-␤-carboline-3-carboxylate; ␤-CCP, propyl-␤-carboline-3-carboxylate; Bmax , maximal binding capacity of a ligand; ␤2L, ␤2 subunit long form; BZ, benzodiazepine; CL 218872, 3-methyl-6-(3-trifluoromethyl-phenyl)-1,8a-dihydro-[1,2,4]triazolo[4,3-b]pyridazine; CNS, central nervous system; ␦+/+ , ␦ subunit wild-type mouse line; ␦−/− , ␦ subunit knockout mouse line; DMCM, methyl-6,7-dimethoxy-4-ethyl-␤-carboline-3-carboxylate; DIS, diazepam-insensitive; DS, diazepam-sensitive; ECxx , concentration producing xx% of maximal enhancement; EDTA, ethylenediaminetetraacetic acid; GABA, ␥-aminobutyric acid; GABAA , ␥-aminobutyric acid type A; GABAB , ␥-aminobutyric acid type B; GABAC , ␥-aminobutyric acid type C; GAD, generalized anxiety disorder; GIS, GABA-insensitive TBPS binding; ␥2S, ␥2 subunit short form; ␥2L, ␥2 subunit long form; HEK, human embryonic kidney; HEPES, N-(2-hydroxyethyl)piperazine-N -(2-ethanesulphonic acid); IC50 , concentration producing half maximal inhibition; KD , equilibrium dissociation constant; Loreclezole, (Z)-1-[2-chloro-2-(2,4-dichlorophenyl)ethenyl]-1,2,4-triazole (R72063); P4S, piperidine-4-sulphonic acid; PET, positron emission tomography; 4-PIOL, 5-(4-piperidyl)isoxazol-3-ol; PKC, protein kinase C; Propofol, 2,6-diisopropylphenol; Ro 15–4513, ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate; Ro 15–1788, ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate (flumazenil); SPECT, single photon emission computer tomography; SR 95531, 2 -(3 -carboxy-2 ,3 -propyl)-3-amino-6-p-methoxyphenylpyrazinium bromide (gabazine); SSC, standard sodium citrate buffer; TBPS, t-butylbicyclophosphorothionate; TBOB, t-butylbicyclo-o-benzoate; THIP, 4,5,6,7-tetrahydroisoazolo[5,4-c]pyridin-3-ol; TM, transmembrane region; Tris, tris(hydroxymethyl)aminomethane ∗ Corresponding author. Tel.: +358-2-333-7542; fax: +358-2-333-7216. E-mail address: [email protected] (E.R. Korpi). 0301-0082/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 1 - 0 0 8 2 ( 0 2 ) 0 0 0 1 3 - 8

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3. GABA binding site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Structural domains of the GABA binding site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Brain regional distribution of GABA binding site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. GABA site alterations as revealed in knockout animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Benzodiazepine binding site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Structural domains for BZ site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. ANT rats as a model system for cerebellar GABAA receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Gene knockout and knock-in models for BZ receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Ion channel binding site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. The [35S]TBPS/picrotoxinin binding site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Coupling of the BZ site to the TBPS site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Coupling of GABA agonist and [35S]TBPS binding sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. GABA-insensitive [35S]TBPS binding sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. Coupling of GABA site antagonist effects to the TBPS site in brain sections. . . . . . . . . . . . . . . . . 5.6. Furosemide interactions with GABAA receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.7. Clozapine interactions with GABAA receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.8. Cations as modulators of GABAA receptor ligand binding and function . . . . . . . . . . . . . . . . . . . . . 5.9. Anticonvulsive compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.10. Effects of ethanol and volatile anesthetics on GABAA receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.11. Structural requirements of the receptor for barbiturates, etomidate, neurosteroids and propofol 6. Possible contributions of rare subunits: γ 1, , θ and π subunits. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Imaging human brain GABAA receptors in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Critical assessment of the significance of ligand binding studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction This introduction aims at introducing the reader to the field of GABAA receptor physiology and pharmacology and is followed by brief reviews of new advances on the heterogeneity and structure of various ligand binding sites. Prototypic GABAA receptors are selectively activated by muscimol, antagonized competitively by bicuculline and non-competitively by picrotoxin. There are many drug binding sites on the GABAA receptor and the most important ones for this review have been schematically depicted in Fig. 1. One of the main messages of this review is to show that, even in the interaction with prototype compounds, native and recombinant receptors display clear heterogeneity. GABAA receptors mediate the bulk of fast inhibitory neurotransmission in the mammalian brain. Inhibition is a fundamental process in brain activity and, therefore, most neuronal brain cells express these receptors on their cell membranes. The brain energy expenditure has been shown to negatively correlate with the activation of GABAA receptors by agonists (Kelly et al., 1986; Ito et al., 1994; Galeffi et al., 2000). This information can be utilized to protect the brain during recovery from serious accidents by pharmacological intervention at these sites. GABAergic mechanisms are directly involved in all physiological and behavioral processes and, indirectly, in many neuropsychiatric illnesses. Although many drugs acting on the ionotropic GABAA

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receptors (Fig. 1) have been clinically useful in the treatment of anxiety, epilepsy, sleep disorders, alcohol withdrawal and in the induction and maintenance of anesthesia, current therapies pose several problems that need to be solved. For instance, in some countries, BZs have become the primary pharmacological treatment for GAD, but treatment of anxiety disorders with BZs is often associated with tolerance development and withdrawal symptoms, which poses the risk of relapse upon discontinuation (Lader, 1995; Ballenger, 2001). Sedative effects and the development of tolerance also generally limit the use of GABAA receptor agonists in the treatment of epilepsy to the short-term medication for status epilepticus (Alldredge and Lowenstein, 1999), although certain BZs are still used for long-term treatment of specific epilepsies, such as myoclonic seizures (Brodie and Dichter, 1996). An important indication for these drugs has been the treatment of insomnia, although the long-term use of BZs as hypnotics also carries the risks of development of tolerance and dependence (Nishino et al., 1998). Daytime drowsiness occurring with long-acting compounds, which may even accumulate in the body with multiple use or rebound insomnia after the use of short-acting drugs, are often reported (Kales et al., 1979). Anterograde amnesia and psychotic states have also been related to the use of some short-acting BZs like triazolam (Kales et al., 1979). Moreover, BZs alter sleep architecture in a typical fashion (Nishino et al., 1998). A problem with anesthesia using GABAA targeting substances, e.g. volatile anesthetics, has been the titration of efficacy in such a way that patients remain stably unconscious

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Fig. 1. Schematic representation of a GABAA receptor with some of the ligand binding sites discussed in the review. It is cut open to expose the internal anion channel, displaying three of the five subunits. When the receptor is stimulated, it allows anions, mostly chloride, to pass along the electrochemical gradient, in mature neurons causing hyperpolarization of the post-synaptic membrane. The GABA, e.g. muscimol, bicuculline and 4-PIOL, and benzodiazepine, e.g. diazepam, flumazenil and DMCM, recognition sites are known to be extracellular, whereas several other sites, e.g. barbiturates, neurosteroids, and alcohol, may be mostly at the TM regions. Zn2+ , La3+ , picrotoxinin and TBPS most likely bind to sites in the channel proper. The loreclezole and the non-BZ agonistic DMCM sites are still undefined.

during operations, which can most likely be achieved, if the exact mechanisms and brain locations of the anesthetic action would be known and could selectively be activated. Two decades ago, attempts were made to solve the problems encountered with the usage of GABAA receptor agonists by using compounds with partial agonist activities at the receptors. However, in spite of their efficacy in animal models, results of clinical trials in patients with anxiety disorders, such as GAD are not truly convincing, neither with regard to efficacy nor with regard to side effects (Pollack et al., 1997; Rickels et al., 2000). But the molecular biology of GABAA receptors has brought about new hope. The diversity of the receptor far exceeds what researchers expected even while cloning the first subunits (Schofield et al., 1987). The receptor subtypes are formed by different combinations of subunits, whose expression varies in different brain cells and brain regions and whose pharmacological properties also differ, creating an opportunity to target receptor subtypes with novel drugs. Many changes in subunit expression occur in response to drug treatment, e.g. with alcohol (Mhatre and Ticku, 1993; Grobin et al., 1998), barbiturates (Tseng et al., 1993; Ito et al., 1996; Lin and Wang, 1996), neurosteroids (Smith et al., 1998; Grobin and Morrow, 2000) and BZs (Holt et al., 1996; Impagnatiello et al., 1996; Pesold et al., 1997; Liu and Glowa, 1999). Therefore, the relative density of receptor subtypes and their functional efficacies in the brain adapts to a changing internal environment. It is still a great challenge to investigate how the receptor subtypes are regulated during acute and chronic drug treatments and how the

knowledge gained can be utilized to prevent tolerance and dependence. Interestingly, no clear dependence and tolerance and only small changes in receptor subunit expression have been reported for BZ partial agonists in experimental animals (Rundfeldt et al., 1995; Holt et al., 1996). However, while the clinical efficacy of these compounds remains to be established (Pollack et al., 1997; Rickels et al., 2000), withdrawal symptoms can be observed after abrupt discontinuation of abecarnil, particularly in patients receiving higher dosages for a longer treatment duration (Pollack et al., 1997). It is presently unknown whether any BZ site ligand, subtype-selective or not, is clinically useful and not causing receptor adaptations in animal model systems. Ligand binding studies on the GABAA receptor have revealed different pharmacological profiles in various brain regions. These profiles can be simulated in vitro using recombinant receptors composed of three different subunits. Several GABAA receptor subtypes differ with respect to their BZ ligand pharmacology, the binding site being largely dependent on ␣␥ subunit combinations (Lüddens and Wisden, 1991). For example, zolpidem has been suggested to be an ␣1 subunit-preferring ligand (Pritchett and Seeburg, 1990), but it has since been observed that zolpidem has no affinity, e.g. to ␣1␤x␥3 receptors (Lüddens et al., 1994). The cerebellar granule cell-specific ␣6 subunit-containing receptors differ in their characteristics of the [3 H]Ro 15–4513 binding site from GABAA receptors elsewhere in the brain (Lüddens et al., 1990; Korpi et al., 1992a; Wong and Skolnick, 1992; Im et al., 1993). This is due to a single amino acid at position 100 in the extracellular domain

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(Wieland et al., 1992; Knoflach et al., 1996) of the ␣6 (and forebrain ␣4) subunit(s) of ␣␤␥ receptors. However, no BZ site ligand has been discovered to selectively act on ␣6 subunit-containing receptors (Section 4.1). Wingrove et al. (1994) have reported that loreclezole, a ligand not acting on the BZ site, recognizes only GABAA receptors that contain ␤2 or ␤3 subunits. In addition, furosemide was found to antagonize selectively ␣6 and ␣4 containing receptors in the presence of ␤2 or ␤3 subunits (Korpi et al., 1995a) (Section 5.6). These examples illustrate the complexity of the structural features that build up a certain ligand binding site and indicate that GABAA receptor classification cannot be based on the contribution of single subunits. These data illustrate the possibility that there are many more ligand binding sites for future drug development on the GABAA receptors than the BZ site. These new targets form a major challenge for research. Small molecules with a subtype-selective action still constitute the prime goal, since it will take more time and effort to utilize gene technology to specifically alter brain neurotransmission. Thus, we focus our review on the known characteristics of several ligand binding sites on the GABAA receptor and summarize possible sites that have a high probability to become interesting structural targets for drug development. It should be kept in mind that at the same time as new therapeutics are being developed, new tools are also emerging to study brain function in general, which led us to as well review the use of GABAA receptor ligands in imaging human brain GABAergic system. We conclude by indicating the shortcomings of the presently existing tools. There are several excellent, broad or focused, reviews on neuronal GABAA receptors, e.g. on structural, functional and pharmacological diversity (Wisden and Seeburg, 1992; Gardner et al., 1993; Kaila, 1994; MacDonald and Olsen, 1994; Rabow et al., 1995; Sieghart, 1995; Stephenson, 1995; Costa and Guidotti, 1996; Lüddens and Korpi, 1996; McKernan and Whiting, 1996; Korpi et al., 1997; Olsen and Avoli, 1997; Sigel and Buhr, 1997; Whiting, 1999; Kneussel and Betz, 2000; Lüscher and Fritschy, 2001; Möhler et al., 2001; Moss and Smart, 2001; Rudolph et al., 2001). We try to avoid repetition of previous publications and describe more recent findings and concepts affecting ligand binding properties both at the native receptor level and following recombinant receptor expression. It should also be noted that GABAA receptors may be expressed in glial cells and possibly serve important functions in their metabolism (Synowitz et al., 2001), a site of GABAA receptor expression we do not discuss in any detail.

2. GABAA receptor basics In the following paragraphs we briefly describe some GABAA receptor features that are not discussed in detail in the main sections. This information is intended

Table 1 Human GABAA receptor subunit genes and gene clusters Chromosome

Subunits

1p36 (mouse 4) 3q11-q13 (mouse ?) 4p12 (mouse 5) 5q33-q34 (mouse ?) 5q34 (mouse 11) 6q14-q21 (mouse 4) 15q11-q13 (mouse 7) Xq28 (mouse X)

␦ ␳3 ␣2, ␣4, ␤1, ␥1 ␲ ␣1, ␣6, ␤2, ␥2 ␳1, ␳2 ␣5, ␤3, ␥3 ␣3, ␪, ε

The chromosomal localizations are from the information submitted to GenBank nucleotide database.

for the novice to the field of GABAA receptor neurobiology. 2.1. Molecular biology GABAA receptor belongs to the superfamily of ligandgated ion channels and is characterized by the heteropentameric structure of the integral anion channel. It was not even 20 years ago, that the purification of bovine GABAA receptors suggested the existence of only two proteins involved in the formation of this ligand-gated Cl− channel (Sigel et al., 1983). Now, it is thought that the GABAA receptors can be composed of a variable array of polypeptide subunits (␣1–6, ␤1–3, ␥1–3, ε, ␦, ␲ and ␪) (Table 1), all of which are products of separate genes (Barnard et al., 1998; Bonnert et al., 1999; Sinkkonen et al., 2000), their variety being extended by the existence of several splice forms, e.g. for ␣6, ␤2 and ␥2 subunits. In addition, the ␳1–3 subunits show similar structural characteristics and sequence homology as the above receptor subunits, but they have been classified as picrotoxinin-sensitive GABAC receptor subunits, based on their pharmacological insensitivity to bicuculline, the prototypic competitive GABAA antagonist (Johnston et al., 1975; Nistri and Sivilotti, 1985). The receptor structures are further altered by post-translational modifications, but the roles of these processes in function and in pharmacological specificity have not been well established. The receptor subunits show sequence similarity of about 70% within classes and about 30% between classes. Their sequence relationships have been visually presented in Fig. 2, together with two more “distant” members of the superfamily, i.e. the nicotinic acetylcholine receptor ␣7 subunit and the glycine receptor ␣1 subunit. Common features for all these subunits as well as for the glycine and nicotinic acetylcholine receptor subunits include four putative TMs and the so-called cysteine loop, located in the N-terminal extracellular domain and characterized by two cysteine residues spaced by thirteen otherwise largely divergent amino acids (Fig. 3). Another classical hallmark of GABAA receptors is a sequence conserved in the second TM encompassing the amino acids TTVLTMTT (Fig. 4). Though this sequence has been used to retrieve 13 of the

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known GABAA receptors (Herb et al., 1992), it is not absolutely conserved in the more recently identified subunits ε, ␲, ␳1–3 and hardly recognizable in ␪. In view of the fact that five of the eight amino acids are proposed to line the ion channel (Xu and Akabas, 1996) (Fig. 4), especially the divergence of the ε and ␪ subunits is rather surprising and warrants an in-depth investigation into the properties of receptors containing these subunits. 2.2. Assembly and membrane targeting

Fig. 2. The phylogenetic tree of GABAA receptor subunit proteins plus the glycine receptor ␣1 and the nicotinic acetylcholine receptor ␣7 subunits as a cladogram. The subunit sequences are those of the rat except the mouse ␪. The data were obtained from public databases and aligned under the HUSAR package using the clustal program. The resulting distance matrix was fed into the program clustree in the HUSAR package. The dendrogram data together with the cluster result were used to generate the cladogram with Phylodendron (http://iubio.bio.indiana.edu). Though all subunits contain the N-terminal cysteine loop (see Fig. 3), i.e. two cysteines spaced by 13 mostly variant amino acids, only the longest known and most abundant GABAA receptor ␣, ␤, ␥ and ␦ subunits contain the conserved ‘TMTT’ motif in the second TM region; the ␪ subunit is the most divergent in that respect, expressing the sequence ‘VLTT’ instead (compare Fig. 4).

Fig. 3. Amino acid comparison of the known GABAA receptor subunits around the cysteine loop (boxed). The consensus sequence in bold shows only those amino acid residues that are identical in all sequences.

The largely but not exclusively heteropentameric assembly of GABAA receptors requires signaling sequences for the specific interaction of the subunits. One of the sequences was identified employing a natural non-assembling splice variant of the ␣6 subunit (Korpi et al., 1994), which was shown to be essential for the assembly of ␣1 and ␣6 subunits with the ␤3 but not with the ␥2 subunit (Taylor et al., 2000). A stretch of 70 amino acids in the second half of the N-terminal extracellular domain was identified to be important for the homooligomeric assembly of the GABAC receptor ␳1 but not the ␳2 subunits (Enz and Cutting, 1999a). In rat ␣1 and ␥2 subunits, domains have been detected [␣1(80–100) and ␥2(91–104)] that are necessary for subunit interaction, assembly and formation of BZ binding site (Klausberger et al., 2000; Klausberger et al., 2001a) in recombinant ␣1␤3␥2 receptors. Another adjacent region of the ␥2 subunit [␥2(83–93)] might be needed for interaction with ␤3 subunits (Klausberger et al., 2001a). Recently, a glia-derived protein was identified in the CNS of a mollusk (Smit et al., 2001). This protein binds acetylcholine, shows sequence similarity to the N-terminus of nicotinic acetylcholine receptor subunits at domains that are suggested to be important in forming the agonist binding sites, and it contains a cysteine loop with 12 (instead of 13) intervening amino acids. It lacks the membrane spanning domains and it forms soluble, i.e. non-membrane-bound and homopentameric complexes (Brejc et al., 2001). This stresses the importance of the extracellular N-terminus for the assembly of subunits in this family of ligand-gated ion channels. Importantly, there are already data to suggest that at least BZ binding sites can be formed by truncated N-terminal extracellular domains in GABAA receptor ␣1 and ␥2 subunit dimers, whereas [3 H]muscimol binding apparently requires transmembrane domains of the ␣1 subunits together with truncated ␤3 subunits (Klausberger et al., 2001a,b). Gephyrin, initially described as a 93 kDa protein co-purified with glycine receptors (Pfeiffer et al., 1982), is now known to be more widely expressed in the CNS as well as peripheral tissue, even in areas devoid of the glycine receptor (Prior et al., 1992). Mice lacking gephyrin die at day 1 after birth (P1) and exhibit a reduced number of clustered glycine receptors at their synapses but not an overall loss of glycine receptors (Feng et al., 1998). As well, a significant reduction in the punctuate immunoreactivity towards the GABAA receptor ␣2 and ␥2 subunits is observed in spinal

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cord sections of these mice (Kneussel et al., 1999). In primary hippocampal neuronal cultures, synaptically clustered GABAA receptors are reduced but their intracellular pool is increased. Together with results from ␥2 knockout mice, which exhibit a loss of clustered GABAA receptors (Craig et al., 1996; Essrich et al., 1998), these data provide evidence for a dominant role for gephyrin and probably the ␥2/3 subunits in GABAA receptor clustering. The pool of GABAA receptors clustered extrasynaptically, as detected by ␤2/3- and ␥2-specific antibodies in wild-type hippocampal neurons, reduces with development, but even after 30 days in culture it amounts to 50% of all clusters (Scotti and Reuter, 2001). This leaves open the question on the mode of specific targeting of these clusters or the precise subunit composition of extrasynaptic versus synaptic clusters. Employing the yeast two-hybrid system a ubiquitously expressed protein, called GABAA receptor-associated protein (GABARAP), was identified to interact with part of the large intracellular loop of the ␥2 subunit (Wang et al., 1999). It exhibits sequence similarity with light chain-3 of microtubule-associated proteins and a putative tubulin-binding motif, which apparently directly interact with microtubules and tubulin, respectively (Wang and Olsen, 2000). Recombinant ␣1␤2␥2L receptors expressed together with GABARAP have variable GABA sensitivity and channel kinetics depending on whether the receptors are in clusters or diffusely distributed on the cell membrane (Chen et al., 2000). Using again the technique of yeast two-hybrid screening, Kanematsu et al. (2002) found an inositol 1,4,5-trisphosphate-binding protein, called p130, that may bind to GABARAP and inhibit the binding of ␥2 subunit to GABARAP. The p130 knockout mice show reduced sensitivity to diazepam both at behavioral and hippocampal eletrophysiological experiments, in the presence of unaltered GABA-induced receptor currents. Another protein, ubiquitin-like protein Plic-1 has been found to interact with several ␣ and ␤ subunits of the GABAA receptor (Bedford et al., 2001). This protein seems to be important for facilitation of GABAA receptor surface expression and intracellular stabilization of subunits. Recently, it was shown that GABAA receptors are constitutively internalized by clathrin-dependent endocytosis (Kittler et al., 2000), which could be traced to the interaction of ␤ and ␥2 subunits with the adaptin complex AP2. This interaction may be functionally important in vivo as blocking the endocytosis increased the amplitude of GABA-induced miniature inhibitory post-synaptic currents in hippocampal neurons by a factor of two. All these studies have witnessed that GABAA receptor subunits cycle between intracellular and plasma membrane compartments with the help of several interacting proteins. The internalization of the ␣1␤2 and ␣1␤2␥2 receptors in HEK 293 cells is strongly modulated by phosphorylation/dephosphorylation reactions (Cinar and Barnes, 2001), but the modulations might not directly depend on any receptor subunit. Especially, the role of PKC-mediated phosphorylation of receptor interacting proteins needs to be

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assessed. The functional and pharmacological regulation of receptor surface expression, subunit stabilization and internalization thus remain to be studied, but the present scarce data already suggests that these mechanisms may also vary between different GABAA receptor subtypes (Moss and Smart, 1996). 2.3. Channel opening Addition of the ligand triggers a small rotation of the extracellular domains of the receptor subunits (Unwin, 1995), which then opens the channel pore formed by the adjoining TM2 regions of the five subunits (predicted from the data obtained with nicotinic acetylcholine receptors; Unwin, 1993). Using disulfide bond mapping in recombinant GABAA ␣1␤1 mutant receptors, Horenstein et al. (2001) could demonstrate that the extracellular portion of the TM2-lined pore (Figs. 4–6) is more flexible than the intracellular portion and that these domains of the ␣1 and ␤1 subunits may rotate asymmetrically, since homologous residues [␣1T261C (compare equivalent position in ␣6 in Fig. 5) and ␤1T256C (compare equivalent position in ␤3 in Fig. 6)] form disulfide bonds only when the receptors are activated by GABA. The resulting covalent modification keeps the channels open. The physical pore properties of GABAA receptors are remarkably invariant among different subunit compositions (Hevers and Lüddens, 1998). Still, different compounds exert their action on the receptor via a range of different modes, e.g. pentobarbital increases the mean duration of opening time and the mean number of openings per burst (Twyman et al., 1989a), but BZ agonists increase the open frequency (Twyman et al., 1989b) and BZ inverse agonists decrease it. Furthermore, though picrotoxinin does not directly interact with the binding site for pentobarbital, it produces the opposite effects on the receptor, i.e. it reduces the mean number of openings per burst and shortens the mean open time (Twyman et al., 1989a). Picrotoxinin protects the covalent modification of an ␣1V257C substitution by a sulfhydryl reagent in the intracellular portion of TM2 region (Xu et al., 1995) (Fig. 4), possibly because of a direct steric hindrance by picrotoxinin. However, it is unresolved, how a direct interaction of picrotoxinin with channel lining residues could exert its effect indirectly, i.e. via the number of bursts and the mean open time instead of changes in the conductance state. Pentobarbital and other barbiturates are unique in the sense that they exhibit three modes of actions on GABAA receptors: at low concentrations they act allosterically on GABA-gated Cl− flux (see earlier), at higher concentrations they open the GABAA receptor channel directly, independent of the presence of GABA. At very high concentrations barbiturates block the Cl− current (Thompson et al., 1996). The former two functions seem to involve the same residues on the ␤ subunits, probably close to the channel-forming TM2 region (Birnir et al., 1997; Serafini et al., 2000). These observations can be cautiously interpreted as to exclude these domains as the direct pharmacophore for barbiturates

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but rather point to their involvement in the transduction machinery, though it is enviable that the two or more independent binding sites on the ␣ subunits of a single receptor in this region display co-operativity, i.e. changing each others affinity once one of them binds a ligand (Yu and Koshland, 2001). As we are still lacking high-affinity ligands for the barbiturate sites, it will take some time and effort to prove or disprove this hypothesis. Opposite to pentobarbital, Zn2+ decreases the on-rate and increases the off-rate (Barberis et al., 2000), thus slowing the onset and accelerating the deactivation kinetics without competitively interacting with the GABA recognition site, again probably involving a different recognition site (see later).

3. GABA binding site 3.1. Structural domains of the GABA binding site The minimal structural requirement for GABAA receptors gated by GABA is a heteropentamer built from two different subunits with one peptide derived from the ␣ class and the other from the ␤ class of variants (Schofield et al., 1987). Thus, it was expected that both subunit classes contribute to the formation of the binding pocket. Indeed, a number of amino acids are members of both classes and most of them, conserved within a subunit class, have been identified as being involved in high-affinity agonist binding. The first one in this series was recognized by the F to L mutation at position 64 in the rat ␣1 subunit (Fig. 5) in an electrophysiological assay (Sigel et al., 1990, 1992), later confirmed by direct photolabeling of the site with [3 H]muscimol (Smith and Olsen, 1994). Whereas the homologous residue in the ␣5 variant was shown to be involved in the formation of the GABA binding pocket, the equivalent residues in the ␤2 and ␥2 subunits do not affect GABA binding (Sigel et al., 1992). The two neighboring amino acids R66, corresponding to R70 in ␣5, and S68 (T in ␣6; Fig. 5) in the ␣1 variant have been reported to contribute to the GABA binding domain (Boileau et al., 1999; Hartvig et al., 2000), as well as R120 in ␣1 (Fig. 5) and its counterpart R123 in ␣5 (Westh-Hansen et al., 1999; Hartvig et al., 2000). The ␣6 subunit is alternatively spliced in approximately 20% of its transcripts in rat brain, causing a 10-amino acid deletion of the amino acids E57 up to Q66, thus including the residues F and R, position 63/65 and 64/66 in ␣6 and ␣1, respectively (Korpi et al., 1994; compare Fig. 5). When this short ␣6 subunit is expressed in HEK 293 cells together with ␤2 and ␥2 subunits, no binding activity is detected. Similarly, when the same subunits are expressed in Xenopus oocytes, no GABA-responsive channels are formed, though the transcript is translated in vitro. Initially, this deletion appears to corroborate with the above idea that the GABA binding domain is at least partly in the extracellular region of the ␣ subunits. Taylor et al. (2000) have, however, shown that the short alternatively spliced ␣6 subunit is never assembled into

receptors that reach the plasma membrane, indicating that the deleted domain and therein most likely Q67 or a subsequent tertiary structural alteration affects membrane targeting. The domains contributing to the GABA binding pocket on the ␤ variants are less clearly defined, though an early study included the two amino acid stretches Y157 to Y160 and T202 to Y205 (Amin and Weiss, 1993; compare Fig. 6). However, recent data substantiating dynamic structural changes of these residues upon binding of GABA and the antagonist SR 95531 (Wagner and Czajkowski, 2001), could be considered as arguments against the direct involvement of the residues T202 to Y205 and should be regarded as elements of the gating machinery according to the standards put forward by O’Shea and Harrison (2000). They tried to directly approach the binding versus the gating sites in the ␣2 subunit by the comparative use of a full agonist (GABA) and a partial agonist (P4S) using the following activation scheme,

where L is the ligand, R the receptor, LR the closed receptor–ligand complex, LR∗ the open ligand-bound channel, k11 and k21 the forward constants for the binding and the gating reactions, respectively, and k21 and k22 the backward constants for the binding and the gating reactions, respectively. Obviously, this scheme is the simplest form and does not take into account ligand-bound closed channels, the requirement for the sequential binding of two ligands or other active or inactive states of the receptor channel. More recently, using substituted cysteine accessibility method on putative ␤-strand structures of the ␤2 subunit with Xenopus oocytes, Boileau et al. (2002) mapped the residues Y97 and L99 as GABA and SR 95531 binding site lining residues, since their modifications were prevented by the presence of the agonist or antagonist. Furthermore, homomeric ␤2(L99C) were found to produce large picrotoxin-sensitive leak currents, much larger than in the homomeric wild-type ␤2 channels. This might indicate that the L99 residue is involved in the allosteric coupling between the GABA binding sites and channel opening mechanisms. 3.2. Brain regional distribution of GABA binding site Early autoradiographic experiments have shown GABAA receptor-associated GABA site labeling using [3 H]muscimol as a ligand (Olsen et al., 1990). More thorough examination has, however, raised the suspicion that the high-affinity binding of GABA site ligands does not distribute as widely as BZ site or channel site ligands. Fig. 7 shows an example of the limited distribution of GABA-displaceable [3 H]muscimol binding in an assay system supposed to yield selective but

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complete GABAA receptor labeling. When the incubation buffer is altered, the binding is slightly increased, but it is still concentrated especially in the cerebellar granule cell layer and thalamus. For example, the diencephalon and colliculi are hardly labeled at all, which contrasts with the labeling patterns for BZ and channel sites (Figs. 9 and 12). These [3 H]muscimol autoradiographies indicate that: (1) the brain regional profile of the GABA sites can be reproducibly determined in various ionic and washing conditions; and (2) that GABA site labeling reveals only a fraction of all GABAA receptors. This is the case though muscimol is the prototype agonist acting on all GABAA receptor subtypes in functional assays at high nanomolar low micromolar concentrations. 3.3. GABA site alterations as revealed in knockout animals

Fig. 7. Representative autoradiographs of high-affinity [3 H]muscimol binding to serial horizontal sections from adult Sprague–Dawley rat brain. Fourteen micrometers cryostat sections were thaw-mounted onto gelatin-coated glass slides, kept frozen at −20 or −70 ◦ C under desiccation for days to months, and then thawed and preincubated three times for 10 min. The incubation in plastic slide mailers or glass jars took 30 min in the dark at ice-bath temperature in the presence of 10 nM [3 H]muscimol, after which the sections were washed either three times for 30 s (normal wash) or three times for 3 s (brief wash). Finally, the sections were dipped into ice-cold distilled water and air-dried at room temperature, and exposed to X-ray film for 5 months. All buffers were adjusted to pH 7.4. The standard buffer contained 0.31 M Tris, buffered with citrate. The experimental buffers were prepared without sodium to prevent any possible GABA transporter binding. They were: (A) 0.17 M Tris buffered with HCl; (B) 50 mM Tris buffered with HCl and supplemented with 120 mM KCl, and (C) 10 mM K-phosphate buffer supplemented with 100 mM KCl. These incubation solutions were used for the preincubation, incubation and washing of the sections. For the images shown, identical tone curves, contrasts and brightness settings were used for all sections from the same film during digitization with an Agfa DuoScan scanner, image processing with Adobe Photoshop 4.0 and the lay-outing with CorelDraw 9.0. The cerebellar granule cell layer (Gr) and thalamus (Th) show strong binding under all conditions, while the mesencephalon (Me), including the inferior colliculi, show only faint labeling or remain practically unlabelled. The hippocampus (Hi), cerebral cortex (Ctx), olfactory bulb (OB) and the striatum (Str) also show consistent labeling. The standard incubation solution produces less labeling than the other non-citrate containing solutions. The short wash left more binding, but no drastic change in the distribution of the labeling was seen as compared to the long wash.

The ␣6 subunit-deficient mice have altered cerebellar GABAA receptor pharmacology in a way that cannot be directly explained by the inactivated subunit (Mäkelä et al., 1997). Therefore, in the ␣6 subunit knockout mouse line the formation of native subunit combinations may not be only a question of the availability of subunits for the assembly with other subunits, but also a question of the structural compatibility of the subunits: the lack of ␣6 subunit causes a strong reduction in the level of the ␦ subunit protein in spite of normal subunit mRNA levels (Jones et al., 1997) (Table 2). The ␣6 subunits are thus critically needed for the ␦ subunits to be assembled into native cerebellar granule cell GABAA receptors, even in the presence of other ␣ subunits. High-affinity [3 H]muscimol binding was proposed to be associated with ␦-containing receptors (Quirk et al., 1995), which is corroborated by the finding that the binding of [3 H]muscimol to GABA sites is reduced in cerebellar sections from ␣6 knockout mice (Mäkelä et al., 1997), which exhibit a reduced number of ␣6 and/or ␦ subunit-containing receptors or may be due to compensatory changes in other receptor subunits in the cerebellum (Uusi-Oukari et al., 2000). By repeating these autoradiographic assays with the forebrains of ␦-deficient mice (Mihalek et al., 1999) it was observed that high-affinity [3 H]muscimol binding to GABA sites is reduced both in the cerebellum and the forebrain of ␦−/− animals, thus further confirming the significance of the ␦ subunit in forming high-affinity GABA sites (Korpi et al., 2002). However, there was no reduction in the number of [3 H]muscimol binding sites in cerebellar membrane homogenates (Tretter et al., 2001), indicating that the autoradiographic images and test tube binding assays represent different populations of GABA binding sites. [3 H]Ro 15–4513 binding to BZ sites of the cerebellum and forebrain was increased in ␦−/− animals, partly due to an increment of diazepam-insensitive receptors indicating augmented assembly of ␥2 subunits with ␣6 and ␣4 subunits (Lüddens et al., 1990; Wisden et al., 1991; Benke et al., 1997). In forebrain membranes of ␦−/− animals, the level of the ␥2 subunit determined by Western blotting

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Table 2 Summary of the GABAA receptor subunit-modified mouse lines Subunit

Modification

Phenotypic notes

Compensation

Reference

␣1

KO, +/−neo

Viable, fertile, no spontaneous seizures >50% reduction in the binding of GABA, BZ and TBPS site ligands in most brain regions due to ␣1␤␥2/3 loss Reduced ␣6 in the cerebellum Strongly reduced GABA responses in Purkinje cells mIPSC amplitudes reduced with prolonged decays in cerebellar stellate neurons and reduced sIPSCs in cerebellar granule cells Normal motor learning and function, slight tremor when handled Normal appearance and drug-free behavior Normal receptor levels, widespread increase in DZ-insensitive BZ binding DZ fails to induce sedation and amnesia, and its anticonvulsant actions are reduced Normal sleep latency, amount of sleep, normal or increased changes in EEG by DZ Normal appearance and drug-free behavior Normal receptor levels DZ fails to induce anxiolysis and myorelaxation at low doses Normal appearance and drug-free behavior Normal receptor levels No known diazepam responses missing, except for high dose myorelaxation

Upregulation of ␣2 and ␣3 subunits depending on the generation

Sur et al. (2001) Vicini et al. (2001)

Not known

Rudolph et al. (1999) McKernan et al. (2000) Tobler et al. (2001)

Not known

Löw et al. (2000) Crestani et al. (2001)

Not known

Löw et al. (2000) Crestani et al. (2001)

No overt neurological phenotype when deleted with ␥3 About 20% decrease in BZ binding and ␤2/3 and ␥2 immunoreactivities due to lack of ␣5 Normal appearance and drug-free behavior Lack of DZ-insensitive BZ binding in the cerebellum; reduction by 20–40% of all binding sites in the forebrain due to interference of ␣1 and ␤2 expression Dramatic loss of ␦ subunit in the cerebellum Enhanced diazepam-induced motor impairment

Not known

Culiat et al. (1994) Fritschy et al. (1997)

Increase in TASK-1 K+ leak channels

Homanics et al. (1997b) Jones et al. (1997) Mäkelä et al. (1997) Korpi et al. (1999) Nusser et al. (1999) Uusi-Oukari et al. (2000) Brickley et al. (2001)

Not known

Sur et al. (2001)

Not known

Homanics et al. (1997a) DeLorey et al. (1998) Krasowski et al. (1998)

KI ␣1(H101R), −neo

␣2

KI ␣2(H101R), −neo

␣3

KI ␣3(H126R), −neo

␣4 ␣5

Not available at present p-Locus deletions

␣6

KO, +neo

␤1 ␤2

Not available at present KO, +/−neo

␤3

KO, +neo

p-Locus deletions

Viable, fertile, no spontaneous seizures, increased locomotor activity >50% reduction in the binding of GABA, BZ and TBPS site ligands in most brain regions due to loss of all main receptor subtypes Slightly reduced GABA responses in Purkinje cells 90% die within 24 h, most have cleft palate Severe loss of GABAA receptors in all brain regions with functional deficits Survivors fertile, but not nursing, hyperactive, hyperresponsive to handling, have motor impairment, learning deficits, epilepsy and in EEG seizure-related abnormalities, show elevated response to cocaine, but no sensitization after repeated administrations Impaired GABA responses in reticular thalamus, but not in relay neurons, leading to synchronized activity Animal model for Angelman syndrome Heterozygous mice show almost normal phenotype Increased amplitudes of ␪ and ␥ band oscillations in olfactory bulbs, with altered olfactory discrimination of different alcohols Cleft palate and neurological phenotype as in ␤3-KO

Huntsman et al. (1999) Resnick et al. (1999) Nusser et al. (2001)

Not known

Culiat et al. (1994) Culiat et al. (1995)

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Table 2 (Continued) Subunit

Modification

␥1 ␥2

Not available at present KO, +neo

␥2L

KI (␥2S ICL), ?neo

␥3 ␦

p-Locus deletions KO, +neo

␲ ε ␪

Not available at present Not available at present Not available at present

Phenotypic notes

Compensation

Reference

Normal embryonic development, postnatal lethality within 3 weeks Sedative and righting responses to DZ are absent Almost all BZ sites disappear, GABA sites present normally, but have small channels kinetically resembling ␣␤ channels Reduced gephyrin-dependent clustering of the receptors, which can be partially rescued by overexpression of ␥3 ␥2+/− mice are viable, showing about 25% reduction in BZ receptors and clustering; these mice have a DZ-sensitive anxious phenotype Only increased ␥2S subunits expressed Normal appearance and drug-free behavior, except for modestly increased anxiety Normal responses to ethanol in behavioral and electrophysiological assays Slightly more sensitive to behavioral actions of BZ agonists coupled with increased receptor affinity to these compounds No overt neurological phenotype Viable, fertile, but produce slightly smaller litters, Normal appearance and drug-free behavior Reduced neurosteroid sensitivity in anxiolytic and anticonvulsant models, unaltered BZ sensitivity Reduced high-affinity GABA site binding and increased DZ-sensitive and -insensitive BZ binding

Not known

Günther et al. (1995) Essrich et al. (1998) Crestani et al. (1999) Lorez et al. (2000)

Not known

Homanics et al. (1999) Quinlan et al. (2000)

Not known Increased ␥2 subunits

Culiat et al. (1994) Mihalek et al. (1999) Tretter et al. (2001) Korpi et al. (2002)

KO, knockout; KI, knock-in; BZ, benzodiazepine; DZ, diazepam; neo, neomycin resistance gene cassette; mIPSC, miniature inhibitory post-synaptic currents; sIPCS, spontaneous inhibitory post-synaptic currents; EEG, electroencephalography; p-locus, pink-eyed dilution locus on mouse chromosome 7; ␥2S ICL, ␥2S subunit intracellular loop.

was increased and that of ␣4 decreased, while the level of the ␣1 subunit remained unchanged (Korpi et al., 2002). The receptor subtype alterations were further confirmed by demonstrating a higher level of the ␣4 subunit that could be co-immunoprecipitated with a ␥2 subunit antibody (Tretter et al., 2001; Korpi et al., 2002). The altered pharmacology of native GABAA receptors and changes in the ␥2 and ␣4 subunit levels in ␦ subunit-deficient mice indicate that the ␦ subunit preferentially assembles in the forebrain with ␣4 subunits, where it interferes with the co-assembly of ␣4 and ␥2 subunits, the ␥2 subunit being recruited into additional functional receptors in its absence. The ␦−/− animals thus constitute the first direct demonstration of a subunit replacement in GABAA receptor knockout mouse lines. The simplest mechanism for the described receptor subtype alterations is based on the availability of various subunits during receptor subunit assembly, i.e. if the ␦ subunit is absent it is replaced by the ␥2 subunit. This poses the question of how this process is regulated in the normal brain. Unfortunately, little is known of GABAA receptor subunit assembly, except that ␣ subunits and especially their N-terminal domains

are obligatory for this process (Tretter et al., 1997; Taylor et al., 2000). Two scenarios exist to explain these results, i.e. either the concentration of ␦ subunit exceeds that of the ␥2 subunit or the ␦ subunit has a higher probability than the ␥2 subunit in assembling with ␣4 and ␣6 subunits. In both scenarios, ␦ and ␥2 subunits compete with each other during assembly into functional receptors in neurons, a process that can efficiently limit the number of receptor subtypes, i.e. subunit combinations, produced. However, we failed to demonstrate mutual exclusion of these subunits in the formation of recombinant ␣1/4/6␤3␥2␦ GABAA receptors in HEK 293 cells (Hevers et al., 2000) where the ␥2 and ␦ subunits assemble into functional receptors as demonstrated by their selective electrophysiological and pharmacological properties, though the expression level is reduced. Further studies on subunit competition in primary neuronal populations are warranted, since neurons, but not fibroblasts, have molecular mechanisms such as clustering proteins (Chen et al., 2000; Kneussel et al., 2000) that might be needed for the selective assembly of subunits. These data illustrate the usefulness of ligand binding techniques in detecting receptor subtype alterations in

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native cells. The data also stresses the differences in native preparations or conditions and recombinant receptor studies: [3 H]muscimol binding to native GABA sites was strongly dependent on the presence of ␦ subunits, while it does not differentiate the high-affinity GABA sites in recombinant receptors which exhibit unaltered binding affinities for this ligand in the presence and absence of the ␦ subunit cells (Hevers et al., 2000). Therefore, [3 H]muscimol autoradiography does not probe all GABA agonist sites with identical efficacy in brain sections, since it seems to work best for ␣6 or ␦ subunit-containing receptors, in a variety of incubation conditions (Fig. 7). Obviously, there is a discrepancy between the high-affinity [3 H]muscimol and [3 H]GABA binding to brain sections, membrane homogenates or recombinant receptor preparations and the low-affinity binding of these ligands suggested from allosteric actions in functional assays employing electrophysiological methods and [35 S]TBPS binding assays. Whereas the former is uniformly in the range of tens of nanomolar (Lüddens et al., 1994), the latter two vary more widely from the high nanomolar to the micromolar range (Lüddens and Korpi, 1995; Hevers and Lüddens, 1998). As this discrepancy holds true for single receptor preparations it cannot be due to receptor heterogeneity but rather must be an intrinsic receptor property. Though the ligand binding assay conditions employed so far on recombinant receptors are not geared to detect any low affinity binding rate, the absence of the high-affinity sites in [35 S]TBPS binding assays and in electrophysiological measurements is even more apparent. One likely explanation is the interconversion between a low-affinity and a high-affinity state, which, however, still awaits a definite biochemical proof. The first experimental data addressing this issue are just appearing in the literature (Bianchi and MacDonald, 2001). The GABA sensitivity of [35 S]TBPS binding in the sections from the ␣6−/− and ␦−/− mouse brains is not so dramatically altered in comparison to wild-type brains as the high-affinity [3 H]muscimol binding itself (Korpi et al., 2002).

4. Benzodiazepine binding site 4.1. Structural domains for BZ site Molecular biological techniques provide powerful tools to examine the structural requirements for any property of a protein. With these methods, it has been possible to detect several critical amino acids in various GABAA receptor subunit classes that affect the affinity, selectivity and intrinsic activity of different BZs. The most important site is in the ␣4 and ␣6 variants, where a single R to H substitution at position 99 and 100, respectively, imparts sensitivity of these receptors to diazepam (Wieland et al., 1992; Wieland and Lüddens, 1994) (Fig. 5). In addition, diazepam insensitivity can be conferred to ␣1 receptors by replacing the corresponding H101 with an R (Wieland et al., 1992). The

point mutation only changes the affinity for diazepam but does not interfere with the affinity for GABA (Kleingoor et al., 1993). Furthermore, diazepam-insensitive ␣6␤2␥2 receptors can be converted to a diazepam-preferring species by four amino acid exchanges in the ␣6 variant, thus reversing the rank order of potency of BZ receptor ligands in mutated as compared to wild-type receptors (Wieland and Lüddens, 1994) (Fig. 5). [3 H]Ro 15–4513 binding to BZ sites is modulated differently by GABA in various recombinant receptors. In the ␣1␤2␥2 receptors, GABA acts as a negative modulator of the binding, which is in agreement with the classification of Ro 15–4513 as an inverse agonist (Bonetti et al., 1989). In the ␣6␤2␥2 receptors, GABA enhances the binding significantly, which concurs with our previous findings on the wild-type ␣6 subunit enriched receptors of cultured cerebellar granule cells and cerebellar homogenates (Malminiemi and Korpi, 1989; Uusi-Oukari and Korpi, 1992). In the mutant ␣6(Q100)␤2␥2 receptor GABA also enhances the binding. [3 H]Ro 15–4513 binding is sensitive to diazepam in ␣1␤2␥2 (K I = 16 nM; Lüddens et al., 1990) and ␣6(Q100)␤2␥2 (K I = 1.3 ␮M) (Korpi et al., 1993) receptors but insensitive in ␣6␤2␥2 receptors. These actions are also consistent with electrophysiological results (Wafford et al., 1993; Knoflach et al., 1996) (but see Kleingoor et al., 1991), demonstrating that inverse agonists act like agonists at ␣6␤x␥2 and ␣4␤x␥2 receptors. A recent study on ␣1H101 mutations by Dunn et al. (1999) supports the idea that the amino acid at this position affects the intramolecular transduction of allosteric effect and not only the ligand binding domain structure, as the intrinsic activities of flumazenil, ranging from antagonistic to partial agonistic and Ro 15–4513, ranging from partial inverse agonistic to partial agonistic, depended on the amino acid replacing the H101. BZ receptor agonist ligands, such as diazepam, CL 218872 and zolpidem, distinguish two GABAA receptor subtypes differing mainly in their ␣ and ␥ subunit variants (Lüddens et al., 1995). They characteristically display a high affinity to the ␣1 subunit-containing receptors, but CL 218872 and zolpidem differ from diazepam in having reduced affinity to ␣2-, ␣3- and ␣5-containing receptors (Pritchett et al., 1989a; Pritchett and Seeburg, 1990). This classification can be extended further, since some ␣␤␥ combinations differentiate between these ligands: zolpidem binds with poor affinity to ␣5 and/or ␥3 subunit-containing receptors (Lüddens et al., 1994), while CL 218872 has 10-fold higher affinity (low nanomolar) towards ␣1␤3␥3 receptors than to any other ␣1 or ␥2/3 subunit-containing receptors. The functional significance of this interaction has not been studied, but it is unlikely that there is a larger pool of receptors with such a high affinity as CL 218872 fails to distinguish any high-affinity components in displacement analysis with rat hippocampal and cerebrocortical receptors (Fig. 8). Similar results can be obtained with autoradiographic experiments, where the maximal displacements of [3 H]Ro 15–4513 binding by diazepam, CL

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Fig. 8. Displacement of the BZ site ligand [3 H]Ro 15–4513 from the flumazenil-sensitive sites of GABAA receptors in rat cerebral cortical and hippocampal membranes by CL 218872 in the presence of zolpidem. The assay was carried out as described in (Uusi-Oukari and Korpi, 1990) with membrane homogenates from the brains of adult male Wistar rats. The concentration of hot ligand was 1 nM, that of unlabelled zolpidem 10 ␮M, and the non-specific binding was defined by the presence of 10 ␮M flumazenil (Ro 15–1788). The concentration of CL 218872 ranged from 0.07 nM to 35 ␮M. The points are means ± S.E.M. for four replications. The displacement curves (drawn through the points) were analyzed by the Prism program: the two-component model gave a better fit (P < 0.01) than the one-component model. The KI values are shown below the figure for both brain regions. They were calculated from IC50 values using the Cheng–Prusoff equation [KI = IC50 /(1 + L/K D )] with L = 1 nM, and K D = 6.4 and 5.4 nM for cerebral cortical and hippocampal receptors, respectively. The specific binding amounted to 141±4 and 152±5 fmol/mg protein, with the zolpidem-insensitive component being 8.9 ± 0.6 and 24.3±0.5%, respectively, for the cortical and hippocampal samples. These results failed to reveal any low-nanomolar affinity sites for CL 218872, e.g. as for recombinant ␣1␤3␥3 receptors, that should be zolpidem-insensitive.

218872 and zolpidem are rather similar, with only small differences in the cortical layers (zolpidem affects less) and in the cerebellar granule cell layer (CL 218872 being more efficient than the others) (Figs. 9 and 10). This is but only one example of the special properties that can be observed in recombinant receptors, but does not seem to exist in appreciable amounts in native brain. It is important to note that the behavioral profiles of diazepam, CL 218872 and zolpidem are quite different and that their behavioral efficacy cannot be deduced from competitive ligand binding assays. Thus, CL 218872 is a low efficacy agonist, diazepam a potent anxiolytic and zolpidem a very potent hypnotic with little anxiolytic efficacy. These differences may be explained by their efficacies: CL 218872 is a partial agonist, diazepam a wide-range full-partial agonist and zolpidem an ␣1 subunit-preferring full agonist (Korpi et al., 1997). Therefore, competitive binding assay

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Fig. 9. Representative mouse brain autoradiographs from an experiment where serial sections were incubated with 5 nM [3 H]Ro 15–4513 for 60 min in the absence and presence of 10 ␮M flumazenil (defining the non-specific binding, producing practically no labeling, not shown), 10 ␮M diazepam, 10 ␮M zolpidem, 1 and 10 ␮M CL 218872. Sections were preincubated in an ice-water bath for 15 min in 50 mM Tris–HCl (pH 7.4) supplemented with 120 mM NaCl, then incubated in similar solution supplemented with the ligand and competing drugs as descibed earlier, and finally washed twice for 30 s in ice-cold incubation buffer, dipped into distilled H2 O, air-dried at room temperature, and exposed to film for up to 6 weeks (Mäkelä et al., 1997). Preparing the images was done as described in Fig. 7, except that for the basal binding, the intensity was scaled down by 50%. The images show that most BZ receptors in the brain are sensitive to diazepam, except for the cerebellar granule cell layer (Gr). There are more zolpidem-insensitive receptors, especially in the hippocampus (Hi), cortical (Ctx) middle and internal layers, and in the caudate/putamen (CPu).

results should be always supplemented with data on the intrinsic efficacies of the compounds before making any behaviorally relevant predictions. The same issue becomes important when one attempts to apply in vitro selectivity data into human brain imaging studies with the purpose to visualize various subtypes of GABAA receptor (Section 7). By exchanging ever smaller regions of the ␣3 subunit sequence with the corresponding portion of the ␣1 subunit, a single G for E substitution, at position 201 was identified by site-directed mutagenesis. The E is conserved in all ␣ variants besides ␣1 (Fig. 5) and its exchange leads to an increase

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Fig. 10. Quantitation of displacement of [3 H]Ro 15–4513 binding from mouse brain sections by various benzodiazepine site ligands. The results are from the experiment shown partially in Fig. 9. Regional labeling intensities of the sagittal mouse brain sections were quantified from the films by using MCID M1 and M4 image analysis devices and programs (Imaging Research, St. Catharines, Canada). Locations of various brain areas on exposed films were identified with the aid of the same brain sections stained with thionin (not shown). Binding densities for each brain area were averaged from measurements from two to three sections. Plastic 3 H-standards (Amersham) exposed simultaneously with the brain sections were used as reference with the resulting binding values given as radioactivity levels estimated for gray matter areas (nCi/mg). The bars are means ± S.D. for four experiments.

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in the affinity for the GABAA receptor subtype-selective compounds CL 218872 and 2-oxo-quazepam (Pritchett and Seeburg, 1991). Amino acids T208 and I215 of the human ␣5 subunit confer high subtype-selectivity of the partial inverse agonist L655708 to ␣5␤␥2 receptors in ␣5(T208S, I215V)␤1␥2 receptors (Casula et al., 2001), the homologous residues affecting also the affinity of zolpidem in ␣1(S208T, V215I)␤1␥2 receptors. However, these residues only slightly reduced the affinity of CL 218872 binding. These effects stress the importance of this domain in BZ binding (Wieland and Lüddens, 1994; Buhr et al., 1997a). In the ␥2 subunit, a single amino acid residue has been identified which seems to critically determine the efficacy of given BZ receptor ligands with GABAA receptor subtypes (Mihic et al., 1994a). If the T142 is converted to serine the channel response of the resulting ␣1␤1␥2 receptor to 5 ␮M GABA is increased by flumazenil and Ro 15–4513 (instead of being unaffected or decreased in the corresponding wild-type receptors), thus converting negative or neutral allosteric modulators into positive ones. The F at position 77 of the ␥2 subunit is homologous to the above-mentioned F64 in the ␣1 and ␣6 subunits (Fig. 5). Whereas the latter is required for high-affinity GABA functionality, the former is involved in high-affinity binding of several BZ receptor ligands, e.g. zolpidem, diazepam and CL 218872 (Buhr et al., 1997b; Wingrove et al., 1997). A similar correspondence was found for amino acids involved in the GABA recognition on the ␤ subunit (Amin and Weiss, 1993) and BZ binding site on the homologous ␣ subunit residues (Amin et al., 1997). Both findings substantiate the claim, that the BZ binding sites is a “converted” agonist recognition site. For some BZ site ligands, such as ␤-carbolines, an additional binding site on GABAA receptors independent of ␥2 subunits has been suggested (Stevenson et al., 1995; Saxena and MacDonald, 1996). In addition to the function as an inverse agonist on the BZ site at low micromolar concentrations, DMCM, ␤-CCE and ␤-CCP at high micromolar concentrations potentiate the GABAA receptor function through a supposedly loreclezole associated binding site in the ␤2 and ␤3 subunits (Wingrove et al., 1994) (Section 5.9). This agonistic effect is independent of the ␣ subunit, and is more pronounced in ␣6-containing receptors due to the lack of inhibition (inverse agonism) by the BZ binding site (Stevenson et al., 1995; Saxena and MacDonald, 1996). This site can be detected especially in the cerebellar granule cell layer by using [35 S]TBPS autoradiography and it is decreased in the absence of ␣6 subunits (Mäkelä et al., 1997). Still another, low-affinity BZ site independent of ␥2 sites has been suggested on the basis of recombinant receptor assays in frog oocytes (Walters et al., 2000). Diazepam, flunitrazepam and midazolam (but not flurazepam) at micromolar concentrations produced a strong enhancement of current induced by a low GABA concentration (EC3 sic!) in ␣1␤2 and ␣1␤2␥2 receptors. Nanomolar concentrations of all the tested BZ agonists normally enhanced GABA responses in the ␣1␤2␥2 receptors, but much less

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than the micromolar concentrations of the active ones and only nanomolar actions were blocked by selective antagonist flumazenil. The homomeric ␳1 receptors do not display the nano- or micromolar BZ potentiation, but the ␳1(I307S) TM2 region-mutants are enhanced by micromolar diazepam. And if the ␣1, ␤2 and ␥2 subunits are converted to ␳1 subunits at the homologous position and the ␣1(S267I)␤2(N265I)␥2(S280I) receptors are expressed, effects by micromolar BZs can no longer be detected. It remains to be shown in native receptors whether these effects have any functional role, the simple test being the demonstration of flumazenil-insensitive sedation or anesthesia. 4.2. ANT rats as a model system for cerebellar GABAA receptors The alcohol-insensitive (AT) and alcohol-sensitive (ANT) rat lines have been developed by selective outbreeding for differential sensitivity to the motor impairing effects of an acutely administered moderate dose (2 g/kg) of ethanol that results in identical blood and brain ethanol levels in these rat lines (Eriksson and Rusi, 1981). The motor impairment is measured with a tilting plane test on a rough surface (Arvola et al., 1958), which evaluates quick postural adaptations, supposedly reflecting cerebellar function. Additionally ANT rats have greater sensitivity to the motor-impairing actions of a number of other drugs: a barbiturate (sodium barbital), an anesthetic (propofol), an N-methyl-d-aspartate receptor antagonist [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten5,10-imine hydrogen maleate (dizocilpine maleate, MK801)] and a neurosteroid agonist (5␤-pregnan-3␣-ol-20-one) (Hellevuo et al., 1989; Toropainen et al., 1997; Yildirim et al., 1997; Korpi et al., 2001). Previous publications showed that the rat lines differ in their sensitivity to motor impairment by lorazepam and diazepam in the tilting plane test (Hellevuo et al., 1989; Wong et al., 1996a). It has also been demonstrated that the motor-impairing effects of ethanol, lorazepam, and barbital were antagonized by picrotoxin (Hellevuo et al., 1989). These results suggest that the involvement of GABAA receptor-mediated mechanisms differ between the rat lines. There are no overall differences in brain GABAA receptors between the ANT and AT rat lines (Malminen and Korpi, 1988; Uusi-Oukari and Korpi, 1992), but the cerebellar receptors are different. The binding of [3 H]Ro 15–4513 to the cerebellum in the ANT rats is about 100-fold more sensitive to diazepam and lorazepam than the binding in the AT rat samples (Uusi-Oukari and Korpi, 1990, 1991), resulting in reduced “diazepam-insensitive” BZ binding in the ANTs. This reduction can be explained by an amino acid change from R to Q in position 100 of the GABAA receptor granule cell-specific ␣6 subunit in the ANT rats, caused by a single point mutation (codon CGA → codon CAA) (Korpi et al., 1993). The mutation could explain the BZ sensitivity of motor reflexes in the ANT rats, since it makes

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the receptors also functionally sensitive to BZ agonists in electrophysiological analyses in HEK cells. [35 S]TBPS binding to the convulsant sites at the GABAA receptors is inhibited by GABA at more than ten-fold lower concentrations in the ␣6␤2␥2 and ␣6(Q100)␤2␥2 receptors than in the ␣1␤2␥2 receptors (Korpi and Seeburg, 1993). Diazepam at 10 ␮M enhances both the stimulation of the binding by low GABA concentrations and the inhibition of the binding by higher GABA concentrations in the ␣1␤2␥2 receptors; it only slightly enhances the inhibition by 100 nM GABA in ␣6(Q100)␤2␥2 receptors, but has no effects in wild-type ␣6␤2␥2 receptors. The mutation might also contribute to the enhanced alcohol, barbiturate, pregnanolone, propofol and MK-801 sensitivity of the ANT rats, but positive proof at the neuronal level for this is missing. The AT and ANT rats differ also in other pharmacological properties of GABAA receptors. The density of high-affinity [3 H]muscimol binding sites is lower in cerebellar membranes of ANT rats than in those of AT rats (Uusi-Oukari and Korpi, 1989; Korpi and Seeburg, 1993; Mäkelä et al., 1996). High-affinity [3 H]muscimol binding is similar in ␣6␤2␥2 and ␣6(Q100)␤2␥2 receptors, with KD values of 3.2 ± 0.4 (mean ± S.E.M., n = 3) and 3.0 ± 0.2 nM, respectively, i.e. in the same range as for ␣1␤2␥2 receptors (K D = 8 nM) (Korpi and Lüddens, 1993), suggesting that the ANT rats have a lower level of ␣6 and/or ␦ subunit-containing receptors, because these subunits are responsible for most of the high-affinity [3 H]muscimol binding in the cerebellum (see earlier) (Korpi et al., 2002). Furosemide antagonism is reduced in cerebellar samples from ANT rats (Mäkelä et al., 1996) and although this correlates with the enhanced diazepam sensitivity of the BZ site, the known ␣6 subunit mutation (Q100) of the ANTs fails to explain the blunted furosemide sensitivity. On the basis of these data, it can be suggested that the ANT rats probably have the most sensitive GABAA receptors in a defined cell population of all living rodents. Electrophysiological experiments on cerebellar sections from these animals, presently being bred at the University of Colorado Health Sciences Center (Dr. Richard Deitrich), are needed to verify the functional characteristics of the intact mutant receptors. Interestingly, even if ANT rats differ from AT rats only in their cerebellar granule cell sensitivity to diazepam, in behavioral tests for the anxiolytic activity of diazepam they show heightened responses as compared to AT rats (Vekovischeva et al., 1999), suggesting that the cerebellum is also important for emotional behaviors. Human studies on the role of ␣6 subunits have suggested that a P385S substitution in the large intracellular domain is associated with reduced diazepam-impairment of smooth pursuit eye movement (Iwata et al., 1999) and reduced alcohol sensitivity and increased development of alcoholism in the offspring of alcoholic fathers (Schuckit et al., 1999). Although the gene cluster on mouse chromosome 11 containing the ␣6 subunit gene may be close to alcohol-related susceptibility genes in quantitative trait loci analyses in mice

(Buck and Finn, 2001), further work is needed to clarify how the neuronal mechanisms of altered cerebellar ␣6 subunit-containing GABAA receptors reduce alcohol sensitivity in behaving animals and apparently in humans. Interestingly, the highly alcohol-sensitive ANT rats consume voluntarily less alcohol than the alcohol-insensitive AT rats (Sarviharju and Korpi, 1993). In proof, Saba et al. (2001) have recently described a Sardinian alcohol non-preferring rat line to spontaneously have the exactly same mutation as the ANT rats in the ␣6 subunit. 4.3. Gene knockout and knock-in models for BZ receptors Recent studies using new gene technological methods have revealed the importance of a few widely expressed subunits, the deficiency of which cannot be compensated for in the developing brain. GABAA receptor ␥2 and ␤3 subunit knockout mouse lines are severely compromised (Günther et al., 1995; Homanics et al., 1997a) and thus these subunits are obligatory at least during development. On the other hand, the lack of ␣5, ␥3, ␥2L, ␣6 and ␦ subunits does not produce any strong behavioral or physiological phenotype (Culiat et al., 1994; Homanics et al., 1997b, 1999; Jones et al., 1997; Mihalek et al., 1999; Collinson et al., 2001). Surprisingly, the deficiencies of ␣1 and ␤2 subunits (Sur et al., 2001), two of the most abundant subunits, are also not associated with lethal or strong behavioral effects, indicating either functional compensation of their lack by other systems and/or a large receptor reserve in the GABAA receptor system. Table 2 summarizes the characteristics of the GABAA receptor-altered mouse lines found and/or produced up to now. Based on the possible significance of the ␣6 subunit in the actions of alcohol (Section 4.2) (Korpi, 1994), it has been of great interest to see how ␣6 subunit-deficient mice would behave and respond to sedative compounds. Two ␣6-deficient mouse lines were independently produced at the same time (Homanics et al., 1997a,b; Jones et al., 1997) and they both lacked the BZ agonist-insensitive [3 H]Ro 15–4513 binding in the cerebellar granule cell layer. These mice behave normally and they do not exhibit any motor learning or co-ordination deficits. Their alcohol and anesthetic sensitivity is not different from that of wild-type animals (Homanics et al., 1998), but their motor function is more readily affected by diazepam than that of the wild-type mice (Korpi et al., 1999). The most interesting findings obtained from these mice come from neurochemical experiments and have been described earlier (Section 3.3): the ␣6 knockout animals are also losing ␦ subunit protein in the presence of normal mRNA expression (Jones et al., 1997), thus strongly affecting the cerebellar [3 H]muscimol binding. Curiously, ␣6 subunit gene manipulations also affect the transcription of neighboring genes, i.e. the expression of ␣1 and ␤2 subunits are reduced especially in the forebrain regions where ␣6 subunit is not expressed (Uusi-Oukari et al., 2000). A likely reason for the inter-

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ference is the presence of the neomycin resistance gene in the gene cluster (Table 2). This data indicates that the loss of about 20–50% of various ␣1 and ␤2 subunit-containing receptor subtypes does not affect normal behavior and, as well, corresponds with the more recent knockout studies on ␣1 and ␤2 subunits (Sur et al., 2001). They, as well, found evidence for another subunit interaction in ␣1 knockouts: the ␣6 subunit-dependent BZ agonist-insensitive [3 H]Ro 15–4513 binding is reduced by about 30%, suggesting that proper assembly or membrane targeting of the ␣6 subunits needs the presence of ␣1 subunits. Further molecular studies are needed to verify the subunit partnerships, since the data presently available on recombinant subunit expression cannot explain the interactions found on the subunit level from these mouse models (Hevers and Lüddens, 2002). A hint to the relevance and the physiological function of the ␣6 subunit is given by the ␣6−/− mice, in which a potassium leak current carried via the Task 1 channel, is increased, probably to counteract the loss of inhibition in the cerebellar granule cells (Brickley et al., 2001). This short summary on ␣6 subunit-deficient mice illustrates the difficulties in predicting the consequences of a missing subunit, since we do not yet understand the rules how the subunits assemble into native receptors. In conclusion, the exact physiological role of the ␣6 subunit-containing GABAA receptors remains unclear, though their presence is diminishing the motor impairment that BZ site agonists can produce. Möhler and co-workers have utilized a gene knock-in approach to alter the BZ-sensitive sites in ␣1, ␣2 and ␣3 subunits into insensitive ones by changing the histidine residue to arginine residue at the same critical site in the extracellular ligand binding domain where the ␣6 subunit of ANT rats is mutated. Given the differential expression levels and brain regional patterns, these novel mouse models have produced important information on the behavioral roles of GABAA receptor subtypes: ␣1(H101R) mutants lose their sensitivity to diazepam-induced sedation and amnesia, partially to diazepam-induced anticonvulsant effects, while retaining normal anxiolytic responses (Rudolph et al., 1999). These animals have normal effects of diazepam on sleep-related parameters (Tobler et al., 2001), suggesting that sedation and sleep-induction can be separated in the involvement of GABAA receptor subtypes. This same mutation has been independently produced by McKernan et al. (2000) and they found very similar behavioral profiles as Rudolph et al. (1999). However, possibly due to differences in testing procedures, their ␣1(H101R) mutants displayed a strong locomotor activation in response to diazepam. The ␣2(H101R) mice displayed no clear anxiolytic responses to diazepam, while their sedative responses to BZs were normal (Löw et al., 2000). Besides the reduction in the high dose diazepam actions on muscle relaxation the ␣3(H126R) mice displayed no defects in the actions of diazepam (Crestani et al., 2001), though the ␣3 subunit is strategically expressed in important monoaminergic nuclei believed to be involved in the regulation of emotional behavior. Interestingly, these

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experiments do not support the idea of an endogenous BZ site agonist, since the basic behavior of the knock-in mice was not altered. Further experiments with these models will be important to exclude compensatory alterations in the GABAergic or other systems and to clarify the roles of these subtypes in behaviors such as tolerance and dependence to BZs. More recently, findings have been presented on the deficiency of the ␣5 subunits, which are largely restricted to the hippocampus: ␣5 subunit-deficient mice show enhanced platform learning in the Morris water maze and normal behavior and normal sensitivity to BZ agonists in the elevated plus-maze test of anxiety (Collinson et al., 2001). It is thus possible that inhibition of ␣5 subunit-containing receptors might help in improving cognitive functions, though the basic function of this subunit can obviously not be to curtail learning ability.

5. Ion channel binding site The [35 S]TBPS binding site is strongly regulated by GABA. All known GABAA receptor antagonists, including bicuculline and SR 95531, inhibit the effects of GABA on these “convulsant” binding sites (Squires et al., 1983; Squires and Saederup, 1987). As GABA reduces the [35 S]TBPS binding and other allosteric agonists facilitate this action in good correlation with the ionophore function determined, e.g. by 36 Cl− flux assays (Edgar and Schwartz, 1990; Im and Blakeman, 1991; Korpi et al., 1995b), it can be used as a biochemical functional assay of GABAA receptors in membrane homogenates and brain sections. Fig. 11 illustrates in a theoretical fashion the drug effects on [35 S]TBPS binding, which should help to understand the following section. 5.1. The [35 S]TBPS/picrotoxinin binding site TBPS and picrotoxinin non-competitively block GABAgated Cl− currents. In a large number of GABAA channels, this blockade is mediated by binding to site(s) that are recognized by several insecticides and anthelmintics, including the cyclodienes lindane and dieldrin, avermectin, the phenylpyrazole fibronil, the active component of absinthe ␣-thujone, as well as picrotoxinin and the 4-propyl-4 -ethynylbicycloorthobenzoate EBOB (Ffrench-Constant et al., 1993; Cole et al., 1995; Hainzl and Casida, 1996; Hold et al., 2000; Ratra and Casida, 2001), though the identity of these sites is not unequivocal (Casida, 1993). The site is often called the convulsant site, the ion channel site or the ionophore site. The first structural information about the protein domain involved in the action of these compounds came from Drosophila melanogaster and D. simulans strains resistant to the insecticidal action of dieldrin, which was traced to an A302S or A302G mutation (Ffrench-Constant et al., 1993) in TM2.

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valine. Only the mammalian ␤ subunits contain an alanine at this position of TM2. The importance of this alanine was stressed by the construction of chimeric and point-mutated ␣1␤3 subunit chimeras (Jursky et al., 2000) and by the interaction of the mutant ␣1(V257C) with chemically reactive blockers of the GABAA receptors (Xu et al., 1995; Perret et al., 1999). However, results on the ␳1 and ␳2 variants indicate that high affinity to picrotoxinin can be achieved by a serine at this position as well, implying that there is a relative, but not an absolute requirement for alanine in TM2. This conclusion is supported by results obtained with the ␣1␤3 chimera point-mutated in a number of positions: interpreting the results conservatively, this study questions the involvement of A302 of the D. melanogaster GABAA receptor or its equivalents in the binding pocket of TBPS as opposed to a more remote effect on the geometry of the channel pore. 5.2. Coupling of the BZ site to the TBPS site

Fig. 11. Representative alterations of [35 S]TBPS binding to well-washed rat cerebrocortical (A) and cerebellar (B) membranes induced by various GABAA receptor ligands. Shown are the responses relative to control, set to 100% (solid line). All drug responses were obtained at externally added GABA (G, here 3 ␮M, unless otherwise stated), which results in cortical membranes in a ca. 60% inhibition (dashed line) and in cerebellar membranes in a ca. 80% inhibition (dashed line). The GABAA antagonist bicuculline alone at 30 ␮M counteracts any action of residual GABA, thus slightly increasing the [35 S]TBPS binding above control values. Furosemide does not act on cortical membranes at the concentration of 100 ␮M, but it inhibits the action of GABA on the cerebellar membranes. In contrast, diazepam enhances the GABA response, i.e. further inhibits [35 S]TBPS binding, only in cortical membranes. The BZ antagonist flumazenil on its own does not modify the GABA response to any larger extent, but it blocks the diazepam effect in cortical membranes. Ro 15–4513 is an “inverse agonist” only in cortical membranes, thus counteracting the GABA inhibition of the binding, whereas it potentiated the GABA effect in cerebellar membranes. Both actions are reversed by adding the antagonist flumazenil. The steroid pregnanolone at 1 ␮M does not distinguish between cortical and cerebellar membranes, whereas 100 nM loreclezole enforces GABA more in the cerebellar than cortical membranes. The experiments were carried out as described in (Korpi et al., 1995a), and means ± S.E.M. (n ≥ 3) are given.

This otherwise highly conserved, putatively channel-lining segment (Unwin, 1989) is diverging at this position between the different GABAA and GABAC receptor subunits (Section 2; Fig. 4). The diversity includes the (pseudo)homopentameric ␳1 and ␳2 subunits with a proline and serine at the homologous position, respectively, and extends to all known mammalian ␣ and ␥ variants with a

In spite of the apparent identity of the BZ binding site in rat ␣1␤x␥2 receptors (Pritchett et al., 1989b), the ␤ variants affect the coupling of the BZ site to the [35 S]TBPS binding site. [35 S]TBPS binding is more efficiently enhanced by BZ receptor ligands in ␣1␤1␥2 than in the homologous ␤2 or ␤3-containing receptors (Lüddens et al., 1994). Similar effects were described for bretazenil on human ␣1␤x␥2 receptors, but not for diazepam (Puia et al., 1992). As well, the potentiation of the GABA-gated current and its biochemical equivalent, [35 S]TBPS binding, are dependent on the ␣ variant, as diazepam was less efficacious in ␣5␤3␥2 than in ␣1␤3␥2 receptors to increase [35 S]TBPS binding in the absence of GABA (Lüddens et al., 1994) and to potentiate the GABA-induced Cl− current (Puia et al., 1992). 5.3. Coupling of GABA agonist and [35 S]TBPS binding sites GABA is known to increase [35 S]TBPS binding to ␣1 subunit-containing receptors at lower and to decrease it at higher micromolar concentrations (Korpi and Lüddens, 1993; Lüddens and Korpi, 1995). As well, it is more potent to inhibit [35 S]TBPS binding in the cerebellar granule cell layer than in other brain regions (Korpi et al., 1992b). In addition to the high sensitivity of the cerebellar granule cell layer GABAA receptors, the autoradiographic [35 S]TBPS binding assay reveals only minor GABA sensitivity differences between other rat brain regions. The IC50 ’s for GABA vary only by a factor of two–three between the main brain regions in the rat (Rabe et al., 2000). Rabe et al. (2000) analyzed the effect of GABA on [35 S]TBPS binding and compared it to that of muscimol, the prototypic GABAA selective agonist and its derivative thiomuscimol (Nielsen et al., 1995), as well as to ␤-alanine and taurine, two endogenous amino acids affecting both the glycine and the GABAA receptors (Horikoshi et al.,

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1988). In addition, they employed THIP and P4S, two potent but structurally diverse derivatives that in BZ binding assays have shown partial agonist activity (Braestrup et al., 1979; Karobath and Lippitsch, 1979; Falch et al., 1985), and a compound classified as a low-efficacy partial agonist, 4-PIOL (Krogsgaard-Larsen et al., 1994). As with GABA, little regional variations by the agonistic actions of muscimol, thiomuscimol, THIP, P4S, taurine and ␤-alanine on [35 S]TBPS binding were found (Rabe et al., 2000), and all these ligands diminish [35 S]TBPS binding at high concentration to less than 10% of basal binding, thus behaving as full agonists. All brain areas appeared to be sensitive with only minor quantitative regional variations, indicating no direct correlation with single subunit distributions for any of these compounds. Therefore, the agonistic actions of ␤-alanine and taurine on their own on GABAA receptors in vivo are likely to be very limited. The synthetic agonist THIP differs from the endogenous agonists mentioned above in that its efficacy varies more between brain regions, suggesting that its interaction may partially depend on the receptor subtype. There are several ␣1 subunit-enriched regions, such as globus pallidus, substantia nigra, red nucleus and inferior colliculus (Wisden et al., 1992), where 30 ␮M THIP fails to reduce [35 S]TBPS binding, while in the rostral and caudal ends of the brain the binding is strongly inhibited. The findings by Rabe et al. (2000) corroborate recent observations on recombinant GABAA receptors expressed in Xenopus laevis oocytes (Ebert et al., 1997), where the efficacy of THIP in electrophysiological studies was the same as that of GABA, muscimol and thiomuscimol in ␣2, ␣5 and ␣6 subunit-containing receptors, but clearly lower in ␣1 and ␣3 containing receptors. P4S strongly reduces [35 S]TBPS binding with regional variations rather similar to that observed for the full agonists (Rabe et al., 2000). At a concentration of 10 ␮M, P4S enhances the action of GABA, i.e. it fails to antagonize the action of GABA, resulting in a strong correlation between the effects of P4S and GABA. Though P4S had been described as a partial agonist (Wong and Iversen, 1985), in the [35 S]TBPS binding assay employed there, it acts as a full agonist. The strong inhibition of [35 S]TBPS binding by P4S in all brain regions argues for a full agonistic action of P4S in most or all native receptors, in contrast to Ebert et al. (1994), who found electrophysiological evidence for partial agonism of P4S in recombinant ␣1 subunit-containing receptors. Another presumed partial agonist, 4-PIOL, reduces [35 S]TBPS binding in many brain regions (Rabe et al., 2000). In the forebrain areas, such as the olfactory bulb, cortical regions and hippocampal areas, where 4-PIOL, alone, decreases [35 S]TBPS binding, it fails to potentiate the GABA (2 ␮M) effect, i.e. it displays properties of a partial agonist. Interestingly, the mesencephalic and thalamic regions are not affected at all, even by the saturating concentration of 4-PIOL (300 ␮M). Still, this compound antagonizes the effect of GABA in these brain regions. The cerebellum, especially the granule cell layer, is rather

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insensitive to 4-PIOL, since neither the basal binding nor the GABA action is affected. Thus, 4-PIOL acts like a weak partial agonist or antagonist depending on the brain area equivalent to a receptor subtype dependent pattern of activity. 4-PIOL significantly elevates [35 S]TBPS binding above basal levels in many ␣1 subunit-containing brain regions, a phenomenon characteristic for this receptor subtype at low GABA concentrations (Korpi and Lüddens, 1993) and consistent with low efficacy agonism. Therefore, the failure to show any effect in a brain region rich in ␣6 subunit-containing receptors could be due to masking by the summation of the enhancing and decreasing effects on ␣1 and ␣6 subunit-containing receptors, respectively. In agreement, recombinant ␣1␤2␥2 receptors bind more [35 S]TBPS in the presence than absence of 4-PIOL, while no increase of binding is observed in ␣6␤2␥2 receptors (Rabe et al., 2000). In line with these findings, electrophysiological data reveal clear differences between recombinant receptor subtypes. In the absence of GABA, 4-PIOL acted as a weak agonist in ␣1␤2␥2 receptors by eliciting currents up to 5% of the maximal GABA response, but it failed to evoke any current in ␣6␤2␥2 receptors. Kristiansen et al. (1991) observed that 4-PIOL is unable to produce bursts of GABAA receptor channel openings of long duration in embryonic olfactory bulb neurons, indicating that 4-PIOL fails to induce a proper conducting state of the chloride channel. 4-PIOL reduces GABA-induced current amplitudes in a dose-dependent manner in ␣1- and ␣6-containing receptor isoforms independent of the GABA concentrations used (Rabe et al., 2000), thus exhibiting characteristics of a non-competitive antagonist, though this mode of action needs further clarification by directly addressing this issue. The heterogeneity of GABAA receptors is widely accepted in structural terms. However, by using the functional [35 S]TBPS binding assay, various endogenous agonistic compounds acting on the GABA recognition site fail to display major brain regional differences, suggesting that the structure and function of the GABA agonist sites detected by this technique is rather similar in all GABAA receptor subtypes, at least under in vitro conditions. 5.4. GABA-insensitive [35 S]TBPS binding sites It should be noted that in many recombinant GABAA receptor subunit combinations, [35 S]TBPS binding is not reduced to background levels even by high micromolar GABA concentrations (Lüddens and Korpi, 1995), a phenomenon different from most observations in brain sections (Edgar and Schwartz, 1990; Olsen et al., 1990; Korpi and Lüddens, 1993; Korpi et al., 1996), suggesting that the coupling between agonist and ion channel sites may differ in the two preparations. By optimizing the autoradiographic conditions (Sinkkonen et al., 2001a), a novel pharmacological fingerprint on brain cryostat sections using [35 S]TBPS autoradiography has been detected that might solve this discrepancy. The fingerprint revealed a

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picrotoxinin-sensitive component of [35 S]TBPS binding with a unique brain regional distribution, characterized by its high insensitivity to GABA (Sinkkonen et al., 2001a). The optimization included use of an increased specific radioactivity of [35 S]TBPS and lengthened washing periods of brain cryostat sections (Korpi et al., 1995a; Sinkkonen et al., 2001a), resulting in an improved signal-to-noise ratio. Most of the picrotoxinin-sensitive [35 S]TBPS binding is allosterically displaced by GABA, but the modified conditions reveal brain regions with portions of the binding being practically insensitive to GABA concentrations up to 10 mM. The specific GABAA receptor agonist muscimol as well as taurine and ␤-alanine at 100 mM are similarly ineffective as GABA. Comparable GIS [35 S]TBPS binding is observed in rat, mouse, human and chicken brain sections (Sinkkonen et al., 2001a). The GIS binding is reversible and apparently dependent on Cl− ions as it is not seen under low salt conditions (Sinkkonen et al., 2001a). It is present mainly in the cerebellar granule cell layer, many thalamic nuclei, subiculum and the inner layer of the cerebral cortex, amounting in these brain regions up to 6% of the binding in the absence of GABA. The distribution largely resembles that of the GABAA receptor ␦ subunit, but it was actually enhanced in the mouse brains deficient in this subunit (Sinkkonen et al., 2001a,b). Therefore, the subunit basis of the GIS binding remains unknown, but, as stated above, it can be simulated by a number of recombinant receptor subtypes (Lüddens and Korpi, 1995). Like the GABA-sensitive [35 S]TBPS binding, GIS binding (Sinkkonen et al., 2001a) is strongly decreased by the GABAergic agonists pentobarbital, 5␤-pregnan-3␣-ol-20one, loreclezole and Mg2+ (Majewska et al., 1986; Maksay and Ticku, 1985; Möykkynen et al., 2001; Schwartz et al., 1994; Wingrove et al., 1994). Interestingly, the efficacy of some of these compounds differs between forebrain and cerebellar regions: Mg2+ and the neurosteroid are efficient in the forebrain but not in the cerebellum, while pentobarbital is more efficient in the cerebellar granule cell layer than in the thalamus (Sinkkonen et al., 2001a). These results indicate that the atypical binding is heterogeneous between various brain regions, probably reflecting different properties of receptor subunit combinations in these regions, either at the amino acid sequence or post-translational levels or at differential subcellular targeting. The BZ site ligands are rather inefficient in affecting GIS binding (Sinkkonen et al., 2001a), in keeping with their GABA-dependent allosteric modulatory mode of action, which is detectable particularly at low GABA concentrations. 5.5. Coupling of GABA site antagonist effects to the TBPS site in brain sections The difference in the actions of GABA antagonists detected by [35 S]TBPS ligand autoradiography in brain sections between the cerebellar granule cell and molecular

layers (Korpi et al., 1992b; Korpi and Lüddens, 1993) is due to the sensitivity difference to endogenous GABA between the high-sensitive ␣6 and low-sensitive ␣1 subunit-containing receptor populations. The two GABAA antagonists bicuculline and SR 95531 have further, more subtle brain regional effects (Bristow and Martin, 1988; McCabe et al., 1988; Olsen et al., 1990; Yu and Ho, 1990; Peris et al., 1991; Snead et al., 1992; Bureau and Olsen, 1993; Leeb-Lundberg and Olsen, 1983; Liljequist and Tabakoff, 1993), which could also be due to differences in receptor subunit combinations. At 50 ␮M, GABA almost completely abolishes the [35 S]TBPS binding, which can be fully reversed by bicuculline and SR 95531. However, both bicuculline and SR 95531 at 50 ␮M significantly decrease the binding in most brain regions (Korpi et al., 1996). The exception is again the cerebellar granule cell layer, where the binding is enhanced due to the loss of inhibition by the residual endogenous GABA (Korpi et al., 1992b). Thus, at high concentrations the antagonists decrease the channel ligand binding, which has to be kept in mind when using [35 S]TBPS binding assay as a biochemical functional assay. SR 95531 is a more potent competitive GABA antagonist than bicuculline, i.e. the KI values to inhibit [3 H]muscimol binding are in the middle nanomolar and low micromolar range, respectively (Heaulme et al., 1986; Squires and Saederup, 1987; Yu and Ho, 1990; Ito et al., 1992; Lüddens et al., 1994). Similar differences are described for the interaction of the two ligands as detected by [35 S]TBPS binding (Lüddens and Korpi, 1995). When comparing their effect across whole brain section, some regions, e.g. anterior cingulate and entorhinal cortices, hippocampus, amygdala, and some thalamic and hypothalamic nuclei, stand out, as SR 95531 is strikingly more potent in inhibiting [35 S]TBPS binding than bicuculline (Korpi et al., 1996). This is witnessed by high ratios of bicuculline- to SR 95531-modulated [35 S]TBPS binding. Binding that was equally affected by SR 95531 and bicuculline, occurred prominently in regions with preferential ␣1 mRNA expression, e.g. nucleus of diagonal band, globus pallidus, zona incerta/subthalamic nucleus, pars reticulata of substantia nigra, red nucleus, superior and inferior colliculi, and central periaqueductal gray. The strongest positive correlation between the ratio of bicuculline- to SR 95531-modulated bindings and the subunit levels, estimated on the basis of the regional expression of 13 GABAA subunits in the brain as reported by Wisden et al. (1992) and Laurie et al. (1992), was for the ␣2 subunit (r = 0.709 and 0.672 in the absence and presence of GABA, respectively). Strong positive correlations were also found for the ␣4, ␣5, ␤1 and ␤3 subunits, whereas the ␣1 subunit correlated negatively with the ratios. Table 3 gives updated brain regional estimates for the expression of various subunits based on mRNA data and on immunohistochemical data, providing a basis to test correlations between brain regional pharmacological heterogeneities in rodent brain sections and estimated receptor subunit levels, such as carried out in Korpi et al., 1996. Importantly, this table should not be

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Table 3 Quantitative estimates for brain regional distribution of GABAA receptor subunits in adult rat brain Brain region

GABAA receptor subunit mRNA and protein expression ␣1

␣2

␣3

3 3

1 1 3 2

1 1 3

3 3

3

3

2

Cerebral cortex Layers 1–4 Layers 5–6 Anterior cingulate cortex

2 3 2

2 1 3

1 3 1

2 1 1

Limbic regions, hippocampus, amygdala Entorhinal cortex Subiculum Hippocampus, CA1 Hippocampus, CA 3 Hippocampus, dentate gyrus Bed nucleus stria terminalis, medial Nucleus of horizontal limb of diagonal band Septohippocampal nucleus/teania tecta Lateral septal nuclei Triangular septal nucleus Bed nucleus of anterior commissura Anterior amygdaloid area Amygdala Posteromedial cortical amygdaloid nucleus

2 2 2 1 1 1 3 1 1 1 1 1 1 1

2 2 3 3 3 3

1 1

1 1 2 1 2 1

Basal ganglia/striatum Nucleus accumbens Caudate/putamen Globus pallidus Claustrum Ventral pallidum/substantia innominata

1 1 3 2 3

3 3 1 2

1 3 2 2 2 1 2

Olfactory areas External plexiform layer of olfactory bulb Glomerular layer Internal granular layer Olfactory tubercle Islands of Calleja Primary olfactory cortex

Thalamus, epithalamus Paraventricular thalamic nucleus Anterodorsal thalamic nucleus Centrolateral/medial thalamic nucleus Intermediodorsal thalamic nucleus Lateral posterior/laterodorsal thal. nucleus Ventroposterior thalamic nucleus Zona incerta/subthalamic nucleus Medial habenular nucleus Medial geniculate nucleus Hypothalamus Medial preoptic area/periventricular nucleus Lateral preoptic area Lateral hypothalamic area Anterior hypothalamic area Paraventricular hypothalamic nucleus Ventromedial hypothalamic nucleus Mesencephalon, pons, medulla Substantia nigra, pars reticulata Substantia nigra, pars compacta Ventral tegmental area Interpeduncular nucleus Red nucleus Superior colliculus, superior gray layer Superior colliculus, intermediate gray layer

3 3 2 3 1 3 3

2 1 2 2

␣4

␣5

␥2

␥3

3 3

3 3

1

3

1

1

1 3 3

1 1 1

1 2

2 3 2

2 2 1

3 3 3

2 2 2

1 1

2 1

1 1 3 3 1 1 1 1

2 2 3 3 2 2

2 2 1 1 2 1 3

2 2 3 3 3 1 2 1 2 1

1 1

1 1

1 1

1 1 1

1 1

2 2 3 3 3 2 1 3 1 1

1 1 1

2 2 2

1 2 2

2 1 1

1 1 1

1 1

1 1 3 2 3

3 3

3

1 1 1 1 1

1 1 1 1 1

2 3 3 3 3 3 2

2 1 1

2

2 1 1

1 1 2

2 1

1 1 1 1

1

1 1

2 2

3 1

1 1

2

1

1

1

3 3 2 3 3 3

␣6

␤1

␤2

␤3

2 1

3 3

1

2

1 1 1 1 1 1 1 1

2 2

1 1 2 1

1 2 3 3 1

1 2

3 1 2 3 2 2

1 1 2

3

1

3

3 1 1 3

3 2

1 1

1

1 2 1

3 1

1

1

1 3 2 3

1 1 2

1 2

1

2 1 1 1 1

1 1 2 2

2

1

2

1

1

2

2 1

2 2 1

2 2

3

1 2 1

2

2 1 1 1 1 1 1

␥1

1

1 1 1 2 1 2

1 1 2 1 2 1 1

␦

ε

␪

1 1

1

1 1 1 1

1

1 2 2 1 1

1 3 1 1 3 2 1 1 1

1

3 1

1 1 1 1 1

1 1 1 1 1 1

1 1 1 1

1

1

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Table 3 (Continued) Brain region

Central gray Inferior colliculus Raphe nuclei Locus coeruleus Cerebellum Granule cell layer Molecular layer

GABAA receptor subunit mRNA and protein expression ␣1

␣2

␣3

3 3 2

1 1 1

1

3 3

1

2 2

␣4

␣5

␣6

1

3 1

␤1

␤2

␤3

2 1

1 2 2

1 2

3 1

␥1

␥2

␥3

␦

2 1 2

1 1 2

1 1 1

2 1 1

3

1 2

ε

␪

2 3

2 3

3

The table is based on the published mRNA in situ hybridization data of (Laurie et al., 1992; Persohn et al., 1992; Wisden et al., 1992; Bonnert et al., 1999; Sinkkonen et al., 2000) and subunit protein immunohistochemical data of (Moragues et al., 2000; Pirker et al., 2000; Schwarzer et al., 2001), and as such should not be used to compare the absolute levels of various subunits in any brain region. Thus, the brain regional profile for each subunit is given using the same scale, even if the absolute concentrations of subunit mRNAs or peptides are different. The regional profile of each subunit is presented so that 3 denotes the highest expression of the subunit in question, 2 strong expression, 1 low expression, and no grading (empty fields) very low or undetectable expression. The ␥3 subunit probes have so faint and even staining in many brain regions both in situ hybridization and immunohistochemical assays that we decided to mark its expression with 1. The ␳ subunits are not included, since their expression is relatively low (detected usually only by reverse transcriptase-polymerase chain reaction) even in the highest expressing regions, such as the retina, superior colliculus and pretectal nucleus of the optic tract, cerebellar granule cell layer, cerebral cortex, CA1 region of the hippocampus and spinal cord (Boue-Grabot et al., 1998; Wegelius et al., 1998; Enz and Cutting, 1999b). The ␳2 subunit is the most abundant in the brain. The ␲ subunit has not been included, since it has not been visualized in any brain region up to date. For detailed cellular localization of various major subunits (Fritschy and Möhler, 1995).

used to compare various subunits in quantitative terms; only their regional distributions in the brain can be compared. While testing the pharmacology of a number of recombinant GABAA receptors relevant to the previous outlined study a possible distinction between the antagonist sensitivity and intrinsic modulatory activity of SR 95531 and bicuculline, was suggested (Lüddens and Korpi, 1995), since ␣2␤1␥3 receptors were much more sensitive to the GABA antagonistic effect of SR 95531 than bicuculline, whereas the intrinsic down-modulation of [35 S]TBPS binding by these antagonists was rather similar. Together, both studies have provided an example of the combined use of ligand autoradiographic and recombinant receptor assays to establish novel putative native GABAA receptor subtypes with distinct pharmacological profiles. 5.6. Furosemide interactions with GABAA receptors The loop diuretic furosemide (Greger and Wangemann, 1987) reduces GABA responses in neuronal cell populations by various means. GABAA receptor responses in the cingulate cortex are not immediately affected after furosemide application, but rather after prolonged furosemide perfusion, which interferes with the recovery of Cl− gradients due to the well-described inhibition of Cl− /cation co-transporters (Thompson et al., 1988). In the hippocampal neurons, Misgeld et al. (1986) found no blockade of GABA responses by furosemide, whereas Zhang et al. (1991) observed a blockade by 0.5–1.5 mM furosemide in neurons at an early postnatal period (days 2–5), but not at more mature stages (days 15–20). Furosemide also blocks the potassium chloride co-transporter KCC2 that has been implicated in establishing the chloride ion gradients necessary for the hyperpolarizing action of GABAA receptors in maturing neurons (Rivera et al., 1999). These transporters are devel-

opmentally regulated and are lacking from immature hippocampal neurons, which are still depolarized by GABA. Pearce (1993) has reported two components in the GABAA receptor response in hippocampal slices from adult rats, one of them showing sensitivity to furosemide (0.5 mM). These data suggest that furosemide might act on selected population(s) of central GABAA receptors and, therefore, when testing the effects of furosemide on [35 S]TBPS binding in rat brain sections, it was a surprise to see that the cerebellar granule cell-specific GABAA receptor subtype (␣6␤2/3␥2) was uniquely antagonized by furosemide, but not by bumetanide, another Cl− /cation transporter blocker (Korpi et al., 1995a). Furosemide antagonizes potently, rapidly and reversibly GABA responses of recombinant ␣6␤2/3␥2, but not of ␣6␤1␥2 or the widely-expressed ␣1␤2␥2 receptors in Xenopus oocytes studied with the two-electrode voltage clamp method (Korpi et al., 1995a). [35 S]TBPS binding studies with different ␤ variants in ␣6␤x␥2 receptors showed that furosemide interaction is identical in ␣6␤2␥2 and ␣6␤3␥2 receptors, whereas ␤1-containing receptors are insensitive to furosemide, although the ␣6␤1␥2 receptors exhibit normal inhibition of [35 S]TBPS binding by micromolar concentrations of GABA (Korpi et al., 1995a). Therefore, the structures mediating the furosemide-sensitivity are determined both by ␣6 and ␤2/3 subunits. The specific localization of the furosemide-sensitive [35 S]TBPS binding sites in the brain is thus due to the restricted expression of the ␣6 subunit, since all ␤ subunits are widely expressed in the CNS (see Table 3). The above-cited studies (Zhang et al., 1991; Pearce, 1993) suggest that furosemide-sensitive GABAA receptors occur in hippocampal neurons. Autoradiographic data exclude the presence of any major neuronal group in the rat forebrain directly possessing furosemide-sensitive GABAA receptor channels (Korpi and Lüddens, 1997).

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Furosemide (0.3 mM) significantly increases the affinity, i.e. decreases the KD value by about 40%, of [35 S]TBPS binding to cerebellar, but not to cerebrocortical receptors (Korpi et al., 1995a). There is no effect on the density of binding sites (Bmax ) suggesting that the effect of furosemide reflects conformational alteration(s) in the receptor. This excludes that furosemide competes with [35 S]TBPS for its binding sites. Further studies indicated that furosemide appears to interact with the heterooligomeric receptor complex via a novel recognition site that allosterically regulates the Cl− ionophore. Thus, furosemide is the first subtype-selective GABAA receptor antagonist and should facilitate studies on cerebellar physiology. Even if furosemide would penetrate the blood brain barrier the usage of furosemide in vivo would be complicated by its potent diuretic effects due to the blockade of the Na+ –2Cl− –K+ co-transporter at concentrations similar to those needed to block the cerebellar ␣6␤2/3␥2 GABAA receptor (Schlatter et al., 1983). Still, furosemide may serve as the lead molecule to design novel compounds selectively acting on different GABAA receptor subtypes. When screening structural derivatives of furosemide in the hope to identify molecules with antagonism at cerebellar or cortical GABAA receptors in the absence of Na+ –2Cl− –K+ co-transporter inhibition (Lüddens et al., 1998), we failed to find any strong candidate, but observed with close analogs of furosemide (Fig. 12), two other pharmacological profiles in brain sections: compared to furosemide PF1885 lost the selectivity for the cerebellar granule cell receptors and antagonized GABA effects also in the forebrain, whereas azosemide, the thio-analogue of furosemide, retained the cerebellum-specific antagonism, but in other regions it strongly enhanced the GABA effects on [35 S]TBPS binding. Further studies are needed to test these compounds functionally. Obviously, more structures should be tested as the structure–function relations remain obscure with the present data. The arginine at position 100 in ␣6 subunit (Lüddens et al., 1990) involved in the BZ agonist insensitivity of ␣6-containing receptors (Wieland et al., 1992) can be excluded as a determinative residue in furosemide action as we observed wild-type like furosemide antagonism of GABA inhibition of [35 S]TBPS binding in ␣6(H100)␤2␥2 receptors, and no antagonism in ␣1(H101R)␤2␥2 receptors, i.e. the mutated proteins reacted similarly to the wild-type ␣1-containing receptors (Mäkelä et al., 1996; Korpi and Lüddens, 1997). A 258 bp fragment of the ␣6 subunit is sufficient for normal furosemide inhibition of the GABA-induced current in chimeric ␣1␤3␥2 receptors, whereas ␣6␤3␥2 receptors including this 258-bp fragment from the ␣1 variant are completely insensitive to furosemide (Jackel et al., 1998), suggesting the main structural domain for furosemide antagonism to reside on this domain of the ␣6 subunit. Together with data from Fisher et al. (1997), the primary determinant for furosemide inhibition was identified in a short stretch of amino acids N-terminal to the

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second half of TM1. The responsible amino acid was later shown to be an isoleucine residue (Thompson et al., 1999). A further low potency site not detectable in the presence of the high potency site is located at the carboxy-terminal end of the ␣6 starting with TM2, though the possibility remains that these sites are part of a single binding pocket (Jackel et al., 1998). The identical ␤ subunit specificity of loreclezole, DMCM and furosemide (Wingrove et al., 1994; Stevenson et al., 1995; Thompson et al., 1999) suggested that the same amino acid residue position on the ␤2 and ␤3 subunits favors the binding of the ligands and prevents it in the ␤1 subunit. Indeed, an N to S exchange in the ␤1 subunit was first described to interact with the action of loreclezole (Wingrove et al., 1994) and DMCM (Stevenson et al., 1995) and later extended to furosemide (Thompson et al., 1999). This suggests that this asparagine is not part of the binding pocket of all these ligands, but rather provides for a mechanism for long-range structural modification that these ligands induce. Therefore, the restricted ␣ subunit preference of furosemide excludes the identity of recognition sites for furosemide on one hand and loreclezole/DMCM on the other hand. 5.7. Clozapine interactions with GABAA receptors The atypical antipsychotic clozapine lacks extrapyramidal side-effects but is more often associated with seizures in patients after increased dosage regimens than classical neuroleptics (Pacia and Devinsky, 1994). It has been shown to partially antagonize GABAA receptor binding and function (Squires and Saederup, 1991). In the [35 S]TBPS binding assay, clozapine produces partial antagonism that is obvious in the forebrain regions, but less so in the cerebellum, thereby producing the mirror image of antagonism produced by furosemide (Korpi et al., 1995b). It slightly reduces the binding in the nominal absence of GABA, like some other antagonists (see earlier for other GABAA antagonists), but in a functional 36 Cl− flux assay with forebrain membrane homogenates, it exhibits only antagonism (Korpi et al., 1995b). The clozapine metabolite N-desmethylclozapine, but not clozapine-N-oxide, antagonizes forebrain GABAA receptors at high micromolar concentrations (Wong et al., 1996b). Using recombinant receptors, clozapine has turned out to require opposite structural domains for its antagonism than furosemide: it antagonizes [35 S]TBPS binding to ␣1␤x␥2 and ␣6␤1␥2 receptors but not to ␣6␤2␥2 and ␣6␤3␥2 receptors (Korpi et al., 1995b). Further details of clozapine binding to GABAA receptors remain to be studied, e.g. with mutants that have been found to be important for the antagonism by furosemide. 5.8. Cations as modulators of GABAA receptor ligand binding and function It has been shown that many mono-, bi- and trivalent cations modulate GABAA receptors most likely via several

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Fig. 12. Representative autoradiographs from Wistar rat brains showing the distribution of channel binding sites labeled by [35 S]TBPS in the presence and absence of furosemide and its derivatives with and without 2 ␮M GABA. The structures of the compounds are given on top (Lüddens et al., 1998). Below are the images with 100 ␮M of the drugs. The experiment was carried out using non-equilibrium conditions at room temperature in the presence of 120 mM NaCl, described by Korpi et al. (1995a), modified from (Olsen et al., 1990) and (Edgar and Schwartz, 1990). Fourteen micrometers horizontal sections were cut in a cryostat, thaw-mounted onto gelatin-coated glass slides, dried and frozen. Sections were preincubated in an ice-water bath for 15 min in 50 mM Tris–HCl (pH 7.4) supplemented with 120 mM NaCl. Incubation with [35 S]TBPS (200 dpm/␮l, adjusted to 6 nM with cold TBPS) for 90 min at room temperature (22 ◦ C) was performed in the same buffer, using 600–800 ␮l liquid drops over sections in a humid chamber in the presence and absence of other ligands. After the incubation, the sections were washed three times for 15 s in ice-cold incubation buffer, dipped into distilled H2 O, air-dried at room temperature, and exposed to film for 3–5 days. Twenty micromolar picrotoxinin reduced the signal to background level (not shown). Preparation of the images was done as described in Fig. 7. Azosemide slightly decreases the forebrain binding when used alone, while furosemide and PF1885 do not affect the binding to a large extent, except for increasing the cerebellar granule cell layer (Gr) binding. GABA reduces the binding by increasing the dissociation rate more than the association rate. Furosemide and its analogs antagonize the effect of GABA on the Gr binding. In the forebrain, furosemide does not affect the binding, while azosemide strongly enhances the GABA-induced inhibition and PF1885 antagonizes the GABA-inhibition (increased binding) also in the forebrain. Hi, hippocampus; GP, globus pallidus; Th, thalamus; IC, inferior colliculus; OB, olfactory bulb.

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independent binding sites: intracellular Ca2+ (Inoue et al., 1986), extracellular H+ (Takeuchi and Takeuchi, 1967), Zn2+ (Westbrook and Mayer, 1987), La3+ (Zhu et al., 1998) and Al3+ (Trombley, 1998) all directly modulate GABAA receptor properties. Here, we restrict ourselves to describe the recent developments in the actions of Zn2+ , La3+ and Mg2+ ions, since their mechanisms are partly dependent on certain receptor subunits. The effect of Zn2+ on GABAA receptor responses has been intensively studied. The effects differ depending on the species and maturity of the neurons under study as well as on the subunit composition of the GABAA receptors (Draguhn et al., 1990; Smart et al., 1991). The ␣6␤3␦ GABAA receptor currents are the most sensitive to inhibition by Zn2+ (Burgard et al., 1996; Knoflach et al., 1996; Saxena and MacDonald, 1996), whereas replacement of the ␦ subunit by a ␥ subunit reduces the sensitivity to inhibition by Zn2+ . In addition, substitution of the ␣6 subunit by the ␣1 subunit diminishes it, the rank order of potency of zinc to block GABAA receptors being ␣1␤1␦ > ␣1␤1␥2L␦ > ␣1␤1␥2L (Draguhn et al., 1990; Saxena and MacDonald, 1994). Wooltorton et al. (1997) found that Zn2+ inhibition is greatly reduced by a mutation in TM2 (H292A) in homomeric ␤3 and heteromeric ␣1␤3(H292A) receptors and suggested this residue to be an important determinant of the Zn2+ binding site inside the anion-selective channel of the GABAA receptor (Fig. 6). By using ␥2/␦ subunit chimeras, Nagaya and MacDonald (2001) described a major determinant of Zn2+ insensitivity in the ␥2L subunit in its N-terminal extracellular domain for rat ␣1␤3␥2L receptors. Smaller contributions of the outer orifice of the TM2 (residues S278, T279 and I280) and the extracellular TM2-TM3 loop (residue K285) of the ␥2L subunit were also defined by point-mutated receptors. Also the ␣ subunits in human ␣x␤3␥2L receptors affect the sensitivity and efficacy of Zn2+ inhibition, so that the ␣1 subunits convey higher sensitivity but lower efficacy than the ␣2 or ␣3 subunits (White and Gurley, 1995). All these data indicate that Zn2+ has binding site(s), which is (are) formed by the interaction of various subunits. This is in agreement with Berger et al. (1998) who found that Zn2+ strongly increases the desensitization of hippocampal basket cell GABAA receptors, which were observed to contain members of ␣, ␤ and ␥ subunits. Since enhanced Zn2+ inhibition of the GABAA receptors has been suggested to play a major role in temporal lobe epilepsy (Buhl et al., 1996), it might be worthwhile to apply this molecular information to develop novel inhibitors that block the action of endogenous Zn2+ . Im et al. (1994) observed Zn2+ effects with the [35 S]TBPS binding assay in recombinant receptors. Zn2+ inhibited the binding in the absence of GABA and blocked the GABA-induced inhibition of the binding. Zn2+ should thus reverse the decrease of [35 S]TBPS binding by GABA also in autoradiographic assays, if it behaves as an antagonist. In a study of Mäkelä et al. (1997), the ability of Zn2+ to antagonize endogenous GABA was diminished in cerebellar granule cells of the ␣6−/− mice due to the lack of the

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␣6␦ subunits-containing receptor subtype. However, in the molecular layer of the cerebellum, Zn2+ strongly displaced the binding at 100 ␮M. In agreement, Kume et al. (1994) autoradiographically detected in most brain regions zinc inhibition of [3 H]TBOB binding, a structural analog of [35 S]TBPS. Mg2+ is a less abundant serum electrolyte, but the second most abundant intracellular cation (Elin, 1994) and important for a multitude of metabolic reactions. Mg2+ does not trigger cellular reactions in a Ca2+ -like manner, but small changes in its concentration may act in the fine tuning and co-ordination of cellular activities (Flatman, 1984). Disturbances in Mg2+ metabolism have been detected in many diseases and pathological conditions including alcoholism, premenstrual syndrome, pregnancy eclampsia and chronic fatigue syndrome (Elin, 1994; Johnson, 2001). A well-known neuronal action of Mg2+ is the voltage-dependent blockage of N-methyl-d-aspartate-type glutamate receptors, but it modulates also GABAA receptor function, e.g. by enhancing muscimol-induced 36 Cl− flux into synaptoneurosomes prepared from rat brain (Schwartz et al., 1994) and it inhibits [35 S]TBPS binding to GABAA receptors in brain sections (Oh et al., 1999). In contrast, Im and Pregenzer (1993) found no effect of Mg2+ on [35 S]TBPS binding to cortical synaptosomal membranes at the concentrations of 5 mM. In a recent autoradiographic study, Mg2+ up to 3 mM decreased [35 S]TBPS binding to GABAA receptor ion channels in most brain regions (Möykkynen et al., 2001; Uusi-Oukari et al., 2001), but the effect diminished at 10 mM. A similar biphasic effect of extracellularly applied Mg2+ on GABA-evoked ion currents in recombinant ␣1␤2␥2S, ␣1␤2, ␣2␤2␥2S and ␣2␤2 GABAA receptors expressed in X. laevis oocytes was also observed (Möykkynen et al., 2001), where Mg2+ increased the GABA-induced ion currents at 0.1–1 mM concentrations, but decreased the currents at 10 mM. The results show that physiologically relevant Mg2+ concentrations affect native and recombinant GABAA receptors suggesting physiologically functional Mg2+ binding sites on the complex. The significance and the mechanism of the Mg2+ effect in native neuronal preparations still need exploration. Functional studies have indicated that La3+ , unlike most of the divalent cations, increases GABA-activated currents in native and recombinant GABAA receptors. It does so by increasing the sensitivity of the receptor for GABA (Im et al., 1992; Narahashi et al., 1994). Recombinant GABAA receptor isoforms are sensitive to La3+ in an ␣ subunit subtype-dependent manner. La3+ potentiates ␣1␤3␥2L receptor currents (Im et al., 1992; Saxena et al., 1997), whereas ␣6␤3␥2L currents are weakly and ␣6␤3␦ currents are strongly inhibited (Saxena et al., 1997). The exact molecular mechanisms of La3+ effects are still to be studied. [35 S]TBPS binding to cerebrocortical membranes is stimulated by La3+ via an increase in the affinity for the ligand (Im and Pregenzer, 1993). Interestingly, the inhibition of the binding by micromolar GABA was not enhanced by

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La3+ , although barbiturates and neurosteroids were effective. Thus, [35 S]TBPS binding results are not consistent with the agonistic efficacy of La3+ . However, in autoradiographic assays of [35 S]TBPS binding to cerebellar sections, La3+ also stimulates the binding in the cerebellar granule cell layer, especially in the presence of GABA (Mäkelä et al., 1999; Uusi-Oukari et al., 2001), which is consistent with its antagonistic action on the ␣6␦ subunit-containing receptors, and supported by results in the ␣6 knockout granule cells, where the GABA-antagonistic stimulation of the binding by La3+ was abolished (Mäkelä et al., 1999). 5.9. Anticonvulsive compounds Phenytoin and carbamazepine have been suggested to be GABAA receptor ␣1 subunit-preferring positive allosteric modulators (Granger et al., 1995). However, brain autoradiographic and brain membrane homogenate [35 S]TBPS binding assays did not indicate any clear efficacy for these compounds (Pitkänen et al., 1987; Holopainen et al., 2001a), making it unlikely that these drugs produce anticonvulsant actions via the GABAA receptor. Loreclezole is a broad spectrum anticonvulsant (Wauquier et al., 1990), discriminating between ␤1 and ␤2 or ␤3 subunit-containing GABAA receptors (Wafford et al., 1994). It enhances the GABA-induced activity of ␤2/3-containing GABAA receptors >300 times more than the activity of ␤1-containing receptors. The action of loreclezole depends on the amino acid N290 of the human ␤3 subunits (N289 of the ␤2; Fig. 6), whereas the ␤1 subunit contains a serine in this position (Wingrove et al., 1994). Loreclezole potentiation does not require the presence of a ␥ subunit and also acts in the absence of an ␣ subunit. However, the ␣6, ␥1 and ␥3 subunits, when present, influence the level of potentiation (Wingrove et al., 1994). In spite of this selectivity, an autoradiographic [35 S]TBPS binding assay failed to distinguish the brain regions with different ␤1 to ␤2/3 expression ratios (Holopainen et al., 2001a). Topiramate, a new antiepileptic drug, affects GABAA receptor responses in vitro in some systems and in some neurons (Gordey et al., 2000; White et al., 2000), possibly by interfering with receptor desensitization. However, it also increases the level of GABA in the brain and, therefore, it may use direct and indirect mechanisms to enhance GABAergic neurotransmission. Its effect is not abolished by flumazenil and thus it is not mediated by the primary BZ binding site. Further studies are needed to establish any receptor subtype dependence of topiramate action. 5.10. Effects of ethanol and volatile anesthetics on GABAA receptors Several neuronal signaling systems are sensitive to the acute effects of ethanol at pharmacologically and physiologically relevant concentrations (reviewed in Harris, 1999;

Korpi et al., 1998). Electrophysiological studies on the role of the GABAA receptor have demonstrated brain regional and cellular variation in the susceptibility to the potentiating effect of ethanol, probably due to GABAA receptor heterogeneity (Proctor et al., 1992; Weiner et al., 1997) (reviewed in Grobin et al., 1998). The mechanism of ethanol-induced GABAA receptor enhancement is unclear, and different studies produced controversial results. Potentiation of recombinant GABAA receptors by low concentrations of ethanol has been suggested to depend on the presence of the ␥2L subunit (Harris et al., 1995a; Wafford et al., 1991; Wafford and Whiting, 1992). The additional eight amino acids of the ␥2L variant contain a unique phosphorylation site for PKC in its putative intracellular loop between TM3 and TM4 as compared to the short ␥2S variant (Whiting et al., 1990; Kofuji et al., 1991). One suggestion is that ethanol influences the function of GABAA receptors directly via this phosphorylation site and at higher concentrations via some other site on the receptor (Mihic et al., 1994b). This idea is supported by a study with mutant mice lacking the ␥ isoform of PKC, which show a decreased behavioral sensitivity to ethanol (Harris et al., 1995b). However, several other studies did not find any difference between the ethanol potentiation of GABA responses at recombinant receptors containing the ␥2 short form and the ␥2L isoform (Kurata et al., 1993; Sigel et al., 1993; Mihic et al., 1994b). Criswell et al. (1993) demonstrated a strong correlation between the presence of [3 H]zolpidem binding in specific brain regions, the enhancement of GABAA receptor responses by ethanol and the presence of the ␣1, ␤2 and ␥2 subunits. They failed to see an ethanol enhancement in all those brain regions that contain ␥2L subunit mRNA. Furthermore, Homanics et al. (1999) engineered a mouse line in which all ␥2 subunits are of the short form. The ethanol effects in these mice were similar to control mice, but hypnotic responses to BZs were slightly prolonged (Table 2). These data suggest that the ␥2L isoform is not an obligatory element in ethanol-responsive GABAA receptors. Ethanol does not exert dramatic effects on any of the many binding sites of the GABAA receptor complex (Korpi, 1994). However, the picrotoxinin-sensitive [35 S]TBPS binding has been consistently found to be affected by ethanol in brain membranes, albeit at high concentrations [IC50 about 350 mM; (Cole et al., 1984; Liljequist et al., 1986; Malminen and Korpi, 1988)]. Using recombinant rat ␣1␤2␥2S/L and ␣6␤2␥2S/L receptors expressed in HEK 293 cells (Korpi et al., 1995c), ethanol (10–500 mM) in the absence of added GABA had only minor effects on [35 S]TBPS binding irrespective of the ␥2 splice variant, in line with its above mentioned non-essential role in the ethanol effects. Even at 500 mM ethanol, the inhibition of [35 S]TBPS binding only amounted to 20 and 40% of the basal binding in ␣1␤2␥2S/L and ␣6␤2␥2S/L receptors, respectively, which is less than what has been observed in brain membranes (see above). In the presence of GABA, ethanol (100 mM) decreased the binding in all four subunit combinations again independent

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of the ␥2 splice variant. These “direct” stimulatory and inhibitory effects of ethanol, albeit small, were slightly greater in ␣6␤2␥2 than ␣1␤2␥2 receptors. More recent functional studies clarified the structural requirements of the enhancements by ethanol and volatile anesthetics in GABAA receptors. Mihic et al. (1997) demonstrated by using chimeric constructs consisting of the strychnine-sensitive glycine receptor ␣1 subunit (potentiation by anesthetics) and the GABAA receptor ␳1 subunit (inhibition by anesthetics) that a region of 45 amino acids in the TM2 and TM3 domains is necessary and sufficient for the enhancement of receptor function by alcohols and volatile anesthetics. Two specific amino acid residues in this region of the GABAA and glycine receptors (S270 and A291 in the GABAA receptor ␣1 subunit; Fig. 5) were observed to be critical for these allosteric modulations (Mihic et al., 1997). The same region also controls the “cutoff” observed for the potency of alcohols with increasing hydrocarbon chain length (Wick et al., 1998), which supports the existence of specific alcohol binding pockets. Even more convincing, the S270C mutation in the GABAA receptor ␣1 subunit forms an irreversible recognition site for the alkanethiol anesthetics propanethiol and propyl methanethiosulfonate, that activates receptor function and simultaneously reduces further stimulation by other anesthetics (Mascia et al., 2000). These ethanol-sensitive sites are apparently present also in the homologous TM regions of the GABAA receptor ␣2 and ␤1 subunits, but may not be functional in the ␥2L subunits (Ueno et al., 1999). More detailed experiments with mutants of the ␣2 subunit together with the ␤1 subunit have, in addition to S270 in TM2, identified the amino acids L232 in TM1 and A291 in TM3, which are apparently involved in the binding cavity for the anesthetics isoflurane, halothane and chloroform (Jenkins et al., 2001). In addition, enflurane potentiation of GABA currents in ␣2␤3␥2 receptors are strongly reduced in ␣2␤3(N265M)␥2 and ␣2␤3(M286W)␥2 mutant receptors (Siegwart et al., 2002). This suggests that the binding occurs to a domain with structural contributions from TM1, TM2 and TM3. However, the L232 in TM1 is neither conserved within the ␣ variants nor in the other subunit classes (Figs. 4 and 5). Furthermore, it should be noted that the TM3 is involved in allosteric coupling mechanisms for many ligands. 5.11. Structural requirements of the receptor for barbiturates, etomidate, neurosteroids and propofol Several compounds in use for general anesthesia are strong positive modulators of GABAA receptors. Their receptor subunit and subtype requirements differ from those of volatile anesthetics. Barbiturates, etomidate, neurosteroids and propofol (propofol) both enhance the effects of GABA and activate the receptors directly, i.e. in the absence of GABA. These two actions may require different domains of the receptor subunits.

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Like the anticonvulsant loreclezole, the active enantiomer of etomidate [(R)-(+)-ethyl-1-(1-phenylethyl)-1H-imidazole-5-carboxylate] (Tomlin et al., 1998) can act only if the receptor harbors the ␤2 or ␤3 subunit, but it is inactive in ␤1 subunit-containing receptors (Hill-Venning et al., 1997; Sanna et al., 1997; Cestari et al., 2000; but see Uchida et al., 1995). The action of these compounds is rather independent of the ␣ subunit variant in ␣␤␥ receptors. The same amino acid residue, putatively located in the extracellular half of the TM2 domain of the ␤ subunits, seems to be responsible for etomidate sensitivity and for loreclezole sensitivity: in ␣1/6␤x␥2 receptors, the ␤1(S290N) mutant was sensitive to etomidate’s GABA-enhancing and directly activating effects, while ␤3(N289S) mutants largely lost their sensitivity (Belelli et al., 1997; Moody et al., 1998). The S-containing non-responsive form is dominant in homomeric ␤ subunit-containing receptors, perhaps even if present in a pentameric complex as the sole S290-␤ variant among four N-containing responsive subunits (Cestari et al., 2000). Interestingly, in ␣2␤3(M286W)␥2 receptors the GABA potentiation by loreclezole, but not by etomidate, is lost (Siegwart et al., 2002). Wild-type ␳ subunits are insensitive to barbiturates, but various replacements of W328 in the putative TM3 domain of ␳1 subunits make the homomeric receptors sensitive to pentobarbital (Amin, 1999). Similarly, a ␳1(I314S) mutation in TM2 makes the channels sensitive to pentobarbital, but not to propofol and pregnanolone (Belelli et al., 1999). As discussed above for etomidate, the pentobarbital sensitivity is also dependent on the ␤ subunit TM2 domains, the ␤3(N265S) mutation making the normally pentobarbital-sensitive homomeric receptors insensitive to it, similarly to the less sensitive homomeric ␤1 receptors (Cestari et al., 2000). The difference in the efficacy of direct and GABA-enhancing actions of pentobarbital between receptors with different ␤ variants has not always been detected (Hill-Venning et al., 1997; Sanna et al., 1995a). However, the same site is also important in heteromeric ␣␤␥2 receptors, although the sensitivity differences between ␤1 and ␤2/3 subunit-containing receptors are not as dramatic (Cestari et al., 2000). In addition to the TM2 domains, the glycine residue at the extracellular entrance of TM1 of the ␤2 (and less so of the ␣1) subunit also seems to be important for the enhancing and direct actions of pentobarbital and propofol (Carlson et al., 2000). It is likely that these mutations interfere more with the conformational changes induced by the barbiturates than with their actual binding sites. Studies with ␤3/␳1 subunit chimeras suggested that the pentobarbital binding site is C-terminal from the middle of TM2 (Serafini et al., 2000), but further studies are needed to identify and differentiate the residues forming the binding site and those involved in the gating of the channel. It should be added that in heteromeric receptors the ␣ variant affects the sensitivity for direct activation by barbiturates as well (Fisher et al., 1997), the ␣6 receptors being three times more sensitive than ␣ receptors.

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In vitro binding results using bovine brain membranes suggest that pentobarbital and propofol share a GABA-potentiating mechanism of action (Davies et al., 1998). However, with the help of a mutation in the ␤2 subunit (Y127P), it has been possible to show that the direct action of propofol, but not pentobarbital or etomidate, is absent from ␣1␤2(Y127P)␥2 receptors (Fukami et al., 1999). The mutation saved the modulatory actions of these anesthetics. Sanna et al. (1995b) found no GABAA receptor subtype-selectivity in the GABA-potentiating action of propofol. Pentobarbital can directly activate the GABAA receptor ␣1␤3␥2, ␣1␤3, ␤3␥2 and ␤3 subtypes. Uchida et al. (1997) studied the GABA-independent action of propofol and found an obligatory role for the ␣ subunits. The direct activation by propofol is absent from ␣4␤1␥2 receptors (Wafford et al., 1996), and greater in ␣6␤3␥2 than in ␣1␤3␥2 receptors though propofol shows a higher efficacy in the potentiation of the GABA effect in the latter receptor (Krasowski et al., 1997). However, the IC50 for the inhibition of [35 S]TBPS binding by propofol in ␣-less receptors was only two times greater than in ␣-containing receptor subtypes (Zezula et al., 1996), suggesting that most native GABAA receptor subtypes should show sensitivity to propofol. Carlson et al. (2000) found that the ␣1(G223F) mutation decreases the maximal effect of propofol. In ␣2␤3(N265M)␥2 and ␣2␤3(M286W)␥2 receptors, the GABA potentiating efficacy of propofol is reduced, but only ␣2␤3(N265M)␥2 receptors display reduced direct activation by propofol (Siegwart et al., 2002). The exact binding domain of propofol remains unknown. Neuroactive steroids are naturally occurring steroid metabolites and their synthetic derivatives that modulate brain function by non-genomic mechanisms (Paul and Purdy, 1992). The metabolites are formed locally in brain cells, independent from their peripheral concentrations (Baulieu and Robel, 1990; reviewed in Paul and Purdy, 1992; Lambert et al., 1995). 5␣-Reductase transforms progesterone to 5␣-DPH, which can act on gene transcription via progesterone receptors of brain cells. 5␣-DPH is reduced by 3␣-hydroxysteroid oxidoreductase to allopregnanolone. Allopregnanolone is a potent and efficient agonist at GABAA receptors, being anesthetic on its own (Mok et al., 1991). In different neuronal preparations, distinct neurosteroids have specific binding properties and may act as positive (e.g. allopregnanolone) or negative (e.g. pregnanolone sulfate) modulators of GABA-activated Cl− channels (Majewska et al., 1986; Gee et al., 1988; Peters et al., 1988; Sapp et al., 1992). At high concentrations, neuroactive steroids directly activate GABAA receptor channels (Lambert et al., 1995) recognizing a binding site distinct from those for other GABAA receptor ligands, such as the BZs and barbiturates (Gee et al., 1988; Peters et al., 1988). Although no absolute subunit specificity for neurosteroid agonist modulation of GABAA receptors has been found, it

has been demonstrated that the subunit composition affects their actions (Puia et al., 1990, 1991, 1993; Shingai et al., 1991; Sapp et al., 1992; Korpi and Lüddens, 1993). Steroid potentiation does not depend on the presence of a ␥ subunit, but there is a need for a ␤ subunit (Puia et al., 1990; Hadingham et al., 1993), but not in the manner described, e.g. for etomidate (Hill-Venning et al., 1997; Sanna et al., 1997; Rick et al., 1998), whereas the potency (Belelli et al., 1996) and perhaps the efficacy is modulated by the ␣ subunit (Shingai et al., 1991). In ␣2␤3(M286W)␥2 mutant receptors, the potent neurosteroid agonist alphaxalone induces a robust direct activation, while its GABA-modulating effect is not altered as compared to the wild-type receptor (Siegwart et al., 2002). The expression of the ␦ subunit in recombinant receptors, together with ␣1/6␤3 or ␣1/6␤3␥2 subunit combinations, has been shown to reduce neurosteroid-induced potentiation of GABA-activated currents, although the ␦ subunit-containing receptors could be directly activated by neurosteroids (Zhu et al., 1996). However, Hevers et al. (2000) found that allopregnanolone potentiates rather similarly the GABA responses of all ␣1/6␤␥2/␦ recombinant receptor variants. Neurosteroid agonists and pentobarbital are more efficacious to alter GABA-evoked currents in cells stably expressing ␣4␤3 receptors than ␣4␤3␥2 receptors using a fluorescence resonance energy transfer assay (Adkins et al., 2001). The exact mode of action of neurosteroids is still unknown: in addition to the action on the receptor complex, it is also possible that neurosteroids modify the intracellular metabolism through kinases or their actions are dependent on it, which then indirectly affects the receptor subunits and GABAA receptor sensitivity (Leidenheimer and Chapell, 1997; Brussaard et al., 2000; Fancsik et al., 2000). Obviously, these effects have completely different structural requirements for receptor subtypes, since ligand binding takes place in the extracellular and membrane environment, while the phosphorylation/dephosphorylation occurs intracellularly. GABAA receptor antagonistic neurosteroids (Majewska and Schwartz, 1987) have been studied much less. One of these compounds, [3 H]pregnenolone sulfate, apparently labeled several binding sites on the GABAA receptor (Majewska et al., 1990). These sites differed from those to which the non-sulfated (non-charged) neuroactive steroids bind (Zaman et al., 1992; Park-Chung et al., 1999). Pregnenolone sulfate may decrease the channel open time (Mienville and Vicini, 1989) and/or enhance the GABA-induced desensitization (Zaman et al., 1992; Shen et al., 2000). Its inhibitory action varies little between various GABAA receptor subtypes (Puia et al., 1993), but, deduced from results obtained in recombinant ␣x␤1␥2 receptors, it has been suggested to depend on the ␣(1–3) variants (Zaman et al., 1992). The exact binding site has not been established, but ␣1(V256S)␤2␥2 receptors, mutated in the intracellular end of TM2, show severely affected rates of the pregnenolone sulfate-induced block (Akk et al., 2001).

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6. Possible contributions of rare subunits: ␥1, ⑀, ␪ and ␲ subunits Although ␣, ␤ and ␥ subunit classes doubtlessly are responsible for most of the GABAA receptor-mediated fast inhibition in the mature nervous system, several other subunits may be significant at certain discrete neuronal circuitries. In the following, we give short accounts of possible roles of some of these subunits. The ␥1 subunit is a rarer ␥-variant, still, it seems to be a major subunit in some brain areas, e.g. in the hypothalamus, septum and amygdala (Ymer et al., 1990; Wisden et al., 1992; Araki et al., 1992, 1993; Pirker et al., 2000). Like ␥2 and ␥3 subunits, it forms a BZ recognition site, when co-expressed with an ␣ and a ␤ variant. However, this subunit confers a low affinity to various BZ ligands, including the antagonist flumazenil, as compared to ␣x␤x␥2 subunits (Ymer et al., 1990; Wafford et al., 1993; McKernan et al., 1995), up to the point that the site is no longer detectable by filtration-type binding assays (Lüddens, unpublished data). While zolpidem has some binding affinity, its efficacy as a positive modulator is low in ␥1 subunit-containing receptors (Wafford et al., 1993). The ␥1 subunit is rather abundant, e.g. in the medial preoptic area of hypothalamus (Herbison and Fenelon, 1995), where it can assemble into functional receptors with ␣2 and ␤3 subunits. In this brain region, the ␥1 subunit may be regulated by sex hormones and produce differential pharmacology in male and female rats (Nett et al., 1999), the males having more ␥1 subunit expression and being curiously negatively modulated by zolpidem. While the receptors may also be altered by intracellular modification of various subunits (Brussaard et al., 2000), the selective expression of ␥1 subunits may give a specific signature on the pharmacological features of certain neurons involved in sexually determined properties. The ε and ␪ subunits have fairly similar expression profiles in the rodent brain being especially abundant in the noradrenergic nucleus locus coeruleus in the brainstem (Bonnert et al., 1999; Moragues et al., 2000; Sinkkonen et al., 2000). On the X chromosome, these subunits form a cluster together with the ␣3 subunit gene (Table 1), which is more widely expressed in the brain, being also expressed in the locus coeruleus. The pharmacological features of these subunits have not been worked out in detail, e.g. ␣3␤ε␪ receptors have not been studied to date. It is possible that ε and/or ␪ subunit-containing receptors form important targets for non-BZ GABAA ligands in the down-modulation of locus coeruleus activity, e.g. during anxiety and drug withdrawal (Heikkilä et al., 2001). The ␲ subunit is strongly expressed in the female reproductive organs, but seems to be rare or non-existing in the brain (Hedblom and Kirkness, 1997). It has been demonstrated that the ␲ subunit can assemble in functional recombinant receptors with ␣5, ␤3 and ␥3 subunits, mimicking a possible hippocampal receptor subtype (Neelands and MacDonald, 1999). The resulting ␲ subunit-containing

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receptors were insensitive to BZ with certain specific pharmacological and biophysical characteristics. The existence of functional ␲ subunits in the brain remains to be directly demonstrated. Furthermore, its partners in functional GABAA receptors of the uterus need to be clarified.

7. Imaging human brain GABAA receptors in vivo BZ receptors can be visualized and quantified in vivo with two modern nuclear medicine techniques. While SPECT is able to detect brain uptake and regional enrichment of radiotracers labeled with isotopes that emit photons of low-energy, e.g. 123 I, PET allows to absolutely and dynamically quantify the regional brain distribution of radio-pharmaceuticals labeled with positron-emitting isotopes, such as 11 C or 18 F. The first potent BZ site antagonist flumazenil (Ro 15–1788) was initially labeled with 11 C for use with PET (Hantraye et al., 1984; Maziere et al., 1984; Samson et al., 1985; Shinotoh et al., 1986). The successful labeling of Ro 16–0154 with 123 I ([123 I]iomazenil) for SPECT was described shortly thereafter (Beer et al., 1990). Both compounds display high specific to nonspecific binding ratios with their regional brain uptake representing the known distribution of BZ receptors in the brain (Fig. 13) (Samson et al., 1985; Shinotoh et al., 1986; Holl et al., 1989; Innis et al., 1991a). Their displacement by clinically used BZ agonists like clonazepam, alprazolam and diazepam is rapid and correlates with the in vivo potencies of these compounds (Innis et al., 1991b). The proconvulsant drugs ␤-CCM and DMCM provoke convulsive activity at receptor occupancies between 50 and 80% in occipital and temporal cortices (Hantraye et al., 1987). All these compounds are predicted to reveal little receptor subtype selectivity and, therefore, do not yield any information of specific GABAA receptor BZ site subpopulations. Furthermore, it is not known whether, e.g. flumazenil exhibits the same intrinsic activity as during binding to isolated membranes, i.e. whether it behaves like a neutral antagonist (Möhler et al., 1981), whose binding is not affected by differential activation states of the receptor. However, the volatile anesthetic isoflurane enhances its binding (distribution volume) in vivo in healthy volunteers (Gyulai et al., 2001), suggesting that in human brain [11 C]flumazenil binding is enhanced during activation of GABAA receptors, a feature otherwise described for agonistic ligands. However, this may be due to conformational alteration by the anesthetic rather than an effect via increased GABA agonist site binding. Therefore, further studies are required to understand the functional significance of drug-induced alterations in flumazenil binding in vivo. For the quantitative evaluation of the BZ receptor in PET two and three-compartment models have been proposed to be suitable (Holthoff et al., 1991; Koeppe et al., 1991). A single injection procedure without blood sampling has been suggested recently for quantification of BZ receptor PET

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Fig. 13. Voxel-wise maps of volumes of distribution for [11 C]flumazenil derived by spectral analysis in brain of a human healthy volunteer in sagital (left), transversal (middle), and coronal views (right) (Gründer and Bartenstein, unpublished data).

images (Delforge et al., 1995). Even with SPECT, it is possible to calculate the density (Bmax ) and affinity (KD ) of BZ receptors by using kinetic (Abi-Dargham et al., 1994; Laruelle et al., 1994a) and equilibrium paradigms (Laruelle et al., 1993, 1994b) with comparable results. Alterations of BZ receptor densities have been described in post mortem studies of several neuropsychiatric disorders including Alzheimer’s disease (Shimohama et al., 1988), Huntington’s disease (Walker et al., 1984), and schizophrenia (Kiuchi et al., 1989; Benes et al., 1992). PET or SPECT methodologies have been used to demonstrate alterations in human brain in vivo, most extensively in various forms of epilepsy, but also in psychiatric disorders, such as alcoholism, anxiety disorders, and schizophrenia. For the presurgical evaluation of epilepsies, imaging of BZ and opiate receptors has been most widely applied in addition to studies of cerebral blood flow and glucose metabolism. Results in generalized epilepsy, however, are heterogenous (Duncan, 1997). The BZ receptor density has been reported to be increased in the cerebellar nuclei, while the density in the thalamus was reduced, suggesting a complex alteration of the GABAA –BZ system in this multicausal disease (Savic et al., 1994). Others have reported an even more composite pattern of change in [11 C]flumazenil binding when compared to controls (Duncan, 1997). The number of BZ receptor sites are not changed in childhood and juvenile absence epilepsy (Prevett et al., 1995a). Treatment with valproate was associated with a reduction in [11 C]flumazenil binding (Prevett et al., 1995b). In focal epilepsies, the situation seems to be more obvious. PET and [11 C]flumazenil demonstrated reduced BZ receptor density in epileptic foci of patients with partial epilepsy (Savic et al., 1988). The area of reduced ligand binding in temporal lobe epilepsy is more restricted than is the reduction in glucose metabolism as determined with

[18 F]fluorodeoxyglucose PET (Henry et al., 1993; Savic et al., 1993). In patients with unilateral hippocampal sclerosis, the reduction of [11 C]flumazenil binding is confined to the sclerotic hippocampus and not accompanied by changes in other brain areas (Koepp et al., 1996). Thus, in these particular forms of epilepsies, BZ receptor sites may serve as markers of neuronal integrity, which makes imaging with BZ receptor ligands especially useful, whereas the more extensive glucose hypometabolism in affected brain regions may reflect diaschisis (Duncan, 1997). In patients with Huntington’s disease, [11 C]flumazenil PET revealed a decreased density of BZ receptors in the caudate nucleus even in early disease stages (Holthoff et al., 1993). Others found an inverse relationship between [11 C]flumazenil and D2 -like dopamine receptor binding (as measured with [11 C]raclopride) in the putamen of symptomatic patients. This result was interpreted as reflective of a GABAA receptor upregulation occurring in this disease (Kunig et al., 2000). The sample size of initial studies in alcohol-dependent patients was probably to small to detect alcohol/alcoholismrelated abnormalities in [11 C]flumazenil binding compared to healthy controls (Litton et al., 1993). However, the variance of Bmax values was higher in alcohol-dependent patients. As well, acute alcohol intake was found to have no effect on [11 C]flumazenil binding in the human brain (Pauli et al., 1992). However, in a large patient pool both [11 C]flumazenil and [123 I]iomazenil bindings were reduced in various cortical areas and possibly also in the cerebellum of abstinent alcoholic patients (Abi-Dargham et al., 1998; Lingford-Hughes et al., 1998). Interestingly, these abnormalities could not be detected in a small pool of female patients (Lingford-Hughes et al., 2000). Studies in long-term abstinent alcoholic patients and in subjects at risk for alcoholism are needed to determine whether the reduced numbers of BZ

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receptors play an etiologic role in the pathophysiology of alcoholism or whether these findings are just a result of chronic alcohol toxicity. Furthermore, since detoxification of alcoholic subjects is usually performed with drugs acting themselves on BZ receptors (e.g. with long-acting BZs, such as diazepam), persisting effects of these compounds on receptor numbers cannot be ruled out in recently detoxified subjects. This is exemplified by studies on the BZ effects on brain glucose metabolism in alcohol-dependent patients. While these subjects were originally reported to have a blunted glucose metabolic response to a lorazepam challenge compared to controls, which could reflect reduced BZ numbers in brain of alcoholics, this finding could not be confirmed after sustained abstinence (Volkow et al., 1993, 1997). Several researchers have quantified BZ receptors in anxiety disorders. However, the results are markedly heterogeneous, which might be partly due to the highly variable action of anxiety on cerebral blood flow (Gur et al., 1987). With [123 I]iomazenil-SPECT, it was initially observed that BZ receptor binding is reduced in frontal, temporal and occipital regions of patients with panic disorder compared to patients with epilepsy, suggesting the involvement of the GABAA –BZ receptor complex in panic disorder (Schlegel et al., 1994). A similar generalized reduction of BZ binding sites in panic disorder was found with [11 C]flumazenil-PET (Malizia et al., 1998). But others found a decrease in BZ receptor binding in panic disorder relative to controls only in the left hippocampus and precuneus (Bremner et al., 2000). In GAD, a circumscribed reduction of BZ binding sites in the left temporal lobe has been demonstrated (Tiihonen et al., 1997). Subsequent fractal analysis in this sample revealed a more homogeneous cerebral BZ receptor density distribution in patients with GAD. These authors conclude that high regional heterogeneity of receptor density and other physiological factors are necessary to maintain adaptation ability (Tiihonen et al., 1997). However, other researchers could not detect any significant differences in Bmax , KD or binding potential (Bmax /KD ) in any brain region in patients with anxiety disorders compared to ageand sex-matched healthy controls (Abadie et al., 1999). The exact clinical characterization of large patient samples is required to control for co-morbid disorders such as depression and for previous or even concomitant drug treatment. A dysfunction of GABAergic systems has also been implicated in the pathophysiology of schizophrenia (Benes and Berretta, 2001). However, the results from imaging studies are conflicting, at least as far as the quantification of BZ receptor sites is concerned. Postmortem studies point to an increase in BZ receptor numbers in schizophrenia, but several [123 I]iomazenil-SPECT studies failed to detect such an aberration. Likewise, some authors found [123 I]iomazenil binding to correlate positively with cognitive performance and inversely with positive symptoms (Busatto et al., 1997; Ball et al., 1998), whereas others could detect only minor reductions or no alteration at all (Abi-Dargham et al., 1999; Verhoeff et al., 1999).

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One interesting application for PET studies has been recently achieved from ideas originating from basic research findings of GABAA receptor mutant mice. GABA and GABAA receptors are suggested to be of major importance in brain development and in the pathogenesis of epilepsy (Petroff et al., 1996; Ben-Ari et al., 1997; Olsen and Avoli, 1997). The subunits ␤3, ␣5 and ␥3 map to human chromosome 15q11-q13 within the imprinted Angelman syndrome deletion region (Glatt et al., 1994, 1997; DeLorey and Olsen, 1999). Angelman syndrome is a severe neurodevelopmental disorder with epilepsy and a heterogenous genetic etiology. Recently, a ␤3 subunit knockout mouse line was generated (Homanics et al., 1997a), exhibiting a high early mortality. The survivors have a phenotype with marked similarities to some clinical features of Angelman syndrome patients (DeLorey et al., 1998), supporting the idea that impaired expression of the gabrb3 gene in humans could contribute to manifestations of Angelman syndrome (see Table 2). The ␤3 subunit-deficient mice have a reduced number of GABAA receptors in their brains (Homanics et al., 1997a). The majority of patients have a maternal deletion in chromosome 15q11-q13, which contains the genes for ␤3, ␣5 and ␥3 subunits of the GABAA receptor. Finnish children with Angelman syndrome due either to maternal 15q11-q13 deletion (n = 3) or a mutation in the ubiquitin protein ligase (UBE3A) gene (n = 1), mapping to the same chromosomal region, were studied using [11 C]flumazenil as a tracer (Holopainen et al., 2001b). The binding potential (calculated Bmax /KD ) was significantly lower widely in the brain, including the frontal, parietal, hippocampal and cerebellar regions in AS children with the deletion than in the child with the UBE3A gene mutation. This suggests that the deleted subunits, especially the ␤3 subunit, would be necessary for producing normal densities of GABAA receptors in these patients. Therefore, ␤3 subunit-containing GABAA receptors may play an important role in the brain development during embryonic and neonatal period and also control the normal excitability in the developing and mature brain. Interestingly, the same chromosome 15q11-q13 region may be associated to some forms of autistic disorder (Nurmi et al., 2001) and preliminary evidence suggests that autistic patients show reduced BZ binding in several brain areas in [11 C]flumazenil PET examinations (Pfund et al., 2001) as well as in [3 H]flunitrazepam autoradiography using postmortem brain samples (Blatt et al., 2001). It remains to be studied whether these patients have also GABAA receptor subunit gene deletions, or whether other genes in this region affect brain maturation with secondary deficits in receptor numbers. Clearly, human studies with BZ receptor subtype-selective ligands are needed to further clarify the role of the GABAA –BZ receptor system in neuropsychiatric diseases, such as anxiety disorders, schizophrenia and alcoholism, since the non-selective BZ receptor ligands might not be able to detect distinct functional abnormalities in such a complex neurotransmitter system as the GABAergic. Furthermore, e.g. lorazepam at clinically used doses leads to

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a very small occupancy (<3%) of BZ sites suggesting a considerable receptor reserve (Sybirska et al., 1993). Similarly, the intrinsically very potent hypnotic zolpidem, an ␣1 subunit-preferring ligand, fails to show any selectivity as a displacer, because in humans a pharmacologically relatively high dose of 20 mg occupies not more than 20–30% of BZ receptors labeled with [11 C]flumazenil (Abadie et al., 1996), thus making it difficult to carry out imaging experiments in humans such as shown in Fig. 9 in vitro which visualizes ␣5 subunit-containing receptors. Other ligands used for PET imaging of BZ sites of the GABAA receptors include the partial inverse BZ agonist [11 C]Ro 15–4513 (Halldin et al., 1992) and the cyclopyrrolone derivative [11 C]suriclone (Frost et al., 1986), none of which displays a dramatic subtype selectivity. 2 -[123 I]Iododiazepam and [123 I]NNC 13–8241 have been proposed for use in SPECT (Kuikka et al., 1996; Saji et al., 1993), but they are all subtype-non-selective. Following the description of the synthesis of a fluorinated analogue of flumazenil, 5-(2 -[18 F]fluoroethyl)flumazenil (Moerlein and Perlmutter, 1992), this ligand has recently been characterized in humans (Gründer et al., 2001). Two new, putatively ␣5 subunit-preferring BZ site compounds [methyl-3 H]L655708 and [ethyl-3 H]RY80 have been initially tested as potential ligands, but they failed to preferentially accumulate into the ␣5-rich hippocampus as compared to the ␣5-poor cerebellum and, therefore, they might not be suitable ligands for human brain imaging studies (Opacka-Juffry et al., 1999). Thus, there seems to be a need for the further development of subtype-selective BZ site ligands for human imaging research. Furthermore, BZ site ligands bind only to a portion of GABAA receptors, as not all subtypes do have the BZ site. Many compounds of various structures bind with a high affinity to GABAA receptors, including the ion channel sites, but, unfortunately, the most potent ligands, such as TBOB, are metabolized too quickly and/or are too lipophilic to be useful as ligands for PET imaging (Culbert et al., 1993; Snyder et al., 1995).

8. Critical assessment of the significance of ligand binding studies Table 4 summarizes the minimal subunit requirements for various ligands that bind to GABAA receptor subtypes and indicates the variable nature of requirements and lack of clear data for several ligands. The previous chapters have described the roles of smaller domains or even single residues in the actions of various compounds. However, in vivo it is the receptor subtypes (subunit combinations) that mediate any differential action of a drug. Therefore, in the best case, the knowledge of the exact molecular domains is limited to aid drug developers to design better subtype-selective compounds and to possibly identify molecular causes of neuropsychiatric diseases.

Table 4 Minimal subunit requirements for drug action Ligand

␣i

␤j

␥k

Diazepam CL 218872 (low affinity) CL 218872 (medium affinity) CL 218872 (high affinity) Zolpidem (low) Zolpidem (high) Ro 15–4513 (low) Ro 15–4513 (high) GABA Loreclezole DMCM (non-BZ site) Zn2+ Barbiturates Neurosteroids Ethanol Furosemide TBPS

1–3, 5 2, 3, 5 1 1–3, 5 2, 3 1 1–4, 6 5 1–6 1–6 1–6 1–6 1–6 1–6 ? 6 1–6

1–3 1–3 1–3 1–3 1–3 1–3 1–3 1–3 1–3 2, 3 2, 3 1–3 1–3 1–3 ? 2, 3 1–3

(1), 2, 3 2 2 3 2 2 2, 3 2, 3 – – – – – – ? – –

Listed are the known dependencies on the presence of defined ␣-, ␤- and ␥-variants of some compounds acting on GABAA receptors as determined by ligand binding assays for the BZ, GABA and TBPS. Only differences larger than 10-fold are included. The differences in the EC50 for GABA to open the gate are not listed as they were discussed in detail earlier (Hevers and Lüddens, 1998). A single bar means that the presence of any variant is not required. Not included in the table are single-variant GABAA receptors like homopentameric ␤3 receptors as they are unlikely to play any physiological role, as do any ␣␥ combinations.

In the preceding paragraphs, we demonstrated the usefulness and the mutual supplementation of ligand binding autoradiography of native receptors and in vitro ligand binding assays in recombinant GABAA receptors. However, there is not always such a good correlation between the results on native and recombinant receptors. In this final section, we will critically assess the significance and limits of ligand binding studies by presenting a few problematic cases. GABA site labeling by [3 H]muscimol in autoradiographs obviously fails to visualize all receptor subtypes, with ␣6 and ␦ subunit-containing receptors being detected the easiest (Section 3.3). It is possible that variations of the assay procedure could increase the usefulness of this ligand, but rather large changes in buffer composition and washing steps failed to induce a stronger labeling throughout the brain (Fig. 7). Some questions regarding the high- and low-affinity sites, as discussed in more detail in Section 3.3, remain unresolved, e.g. can all receptor subtypes exist in high- and low-affinity conformations and how are these conformations regulated, what are the exact modalities for the transition(s) and which subunits are involved in the transition properties? The determination of allosteric interactions has revealed some puzzling discrepancies between the assay methods. For example, in an autoradiographic [35 S]TBPS binding method 4-PIOL fails to antagonize the effect of GABA in the cerebellar granule cell layer, i.e. an area expressing ␣1 and ␣6 subunit-containing receptors, whereas in the electrophysiological assay on recombinant ␣1 and ␣6 subunit-containing

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receptors 4-PIOL is an efficient antagonist, the agonistic properties on ␣1 receptors being hardly detectable (Rabe et al., 2000). Like ligand-binding to brain sections and to recombinant receptors, in general, both methods complement each other, the main difference being that the autoradiography detects several receptor subtypes in each brain region, whereas the recombinant systems usually express only a single one. In addition, we still have close to intact cells under electrophysiological conditions allowing rapid phosphorylation and other post-translational protein processing, which is not possible in homogenate binding assays. There may be also some endogenous modulators that may mask certain binding sites, an example being the high GABA sensitivity of the cerebellar ␣6 subunit-containing receptors (Korpi et al., 1992b; Korpi and Lüddens, 1993), which under normal autoradiographic conditions prevents the full labeling of these receptors by [35 S]TBPS. It has been shown that prolonged preincubation of brain sections in the presence of EDTA further reduces the amount of the agonist, which can be seen as increased basal binding of [35 S]TBPS (Mäkelä et al., 1997). Furthermore, [35 S]TBPS binding to brain sections may not be sensitive enough to reveal brain regional pharmacological distributions of heterogeneities dependent on, e.g. ␤ subunit variants, as shown with loreclezole (Holopainen et al., 2001a). This problem may not be due to the assay, but rather due to the low abundance of ␤1 subunit-containing, loreclezole-insensitive receptors, since in recombinant receptors the ␤ subunits affect loreclezole sensitivities similarly in electrophysiological and [35 S]TBPS binding assays (Wingrove et al., 1994; Lüddens, unpublished data). In another study, the GABA inhibition of [35 S]TBPS binding in the mutant ␣6(Q100)␤2␥2 receptors was only marginally enhanced by 10 ␮M diazepam (Korpi and Seeburg, 1993), though a marked diazepam-induced potentiation of GABA currents could be demonstrated in HEK 293 cells expressing the mutant receptor (Korpi et al., 1993). The discrepancy may be caused by the conditions at which the binding was carried out, i.e. it may not have been optimal to detect the allosteric BZ modulation of GABA effects on [35 S]TBPS binding. It is also possible that the BZ-induced conformational changes affect differently the channel gating mechanisms and picrotoxinin binding site. The BZ binding assay usually involves a straight-forward binding process with a very good signal to background ratio, when ligand affinities and binding site distributions are being studied in vitro. BZ site autoradiography is sensitive enough to visualize differences in binding affinities between receptor populations, e.g. in the cerebellum and thalamus (Benke et al., 1997; Mäkelä et al., 1997). However, many other drugs, e.g. ethanol, can produce dramatic changes in the intrinsic activities of BZ site ligands (see Korpi, 1994), in the absence of altered binding properties. Furthermore, it is not clear whether the intrinsic activities are the same across species, the primary problem being the ineffective-

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ness of partial agonists as anxiolytics in human studies without causing sedation (Pollack et al., 1997; Rickels et al., 2000). This question remains to be tested again with newer partial agonists, such as imidazenil (Giusti et al., 1993), which has not been analyzed for anxiolytic activity in humans, but shows a promising profile in monkeys (Auta et al., 2000). In summary, there are conditions under which the methods of ligand autoradiography, homogenate ligand binding and electrophysiology obtain partially divergent results. As long as we do not know the confounding factors, we cannot decide what are the “true” results, e.g. in order to extrapolate them to human brain imaging results. Fortunately, more and more data on regulatory processes like (de)phosphorylation, clustering assembly and cytoskeletal anchoring of GABAA receptors are being published, so that we might be able to solve these problems in the foreseeable future.

Acknowledgements The work was supported by the Academy of Finland (ERK), the Alexander von Humboldt foundation (ERK, HL), the Deutsche Forschungsgemeinschaft (HL, GG) and the Stiftung Rheinland-Pfalz für Innovation (HL).

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