User:Samsara/GABA

The GABA_A receptor is one of the two ligand-gated ion channels responsible for mediating the effects of Gamma-Amino Butyric Acid (GABA), the major inhibitory neurotransmitter in the brain.

Structure and function

The receptor is a multimeric transmembrane receptor that consists of five subunits arranged around a central pore. The receptor sits in the membrane of its neuron at a synapse. The ligand GABA is the endogenous compound that causes this receptor to open; once bound to GABA, the protein receptor changes conformation within the membrane, opening the pore in order to allow chloride ions (Cl^-) to pass down an electrochemical gradient. Because the [reversal potential] for chloride in most neurons is close to or more negative than the resting membrane potential, activation of GABA_A receptors tends to stabilize the resting potential, and can make it more difficult for excitatory neurotransmitters to depolarize the neuron and generate an action potential. The net effect is typically inhibitory, reducing the activity of the neuron. The GABA_A channel opens quickly and thus contributes to the early part of the inhibitory postsynaptic potential (IPSP) (Siegel et al., 1999; Chen et al., 2005).

Subunits

GABA_A receptors are members of the large "Cys-loop" superfamily of evolutionarily related and structurally similar ligand-gated ion channels that also includes nicotinic acetylcholine receptors, glycine receptors, and the 5HT₃ serotonin receptor. There are numerous subunit isoforms for the GABA_A receptor, which determine the receptor’s agonist affinity, chance of opening, conductance, and other properties (Cossart et al., 2005). In man, there are six types of α subunits, three β's, three γ's, as well as a δ, an ε, a π, a θ, and three ρs (Martin and Dunn, 2002; Sieghart et al., Neurochem Int 1999;34:379–85). Five subunits can combine in different ways to form GABA_A channels, but the most common type in the brain has two α's, two β's, and a γ (Martin and Dunn, 2002). The receptor binds two GABA molecules (Siegel et al., 1999; Colquhoun and Sivilotti, 2004), somewhere between an α and a β subunit (Martin and Dunn, 2002).

Agonists and antagonists

Other ligands (besides GABA) interact with the GABA_A receptor to activate it (agonists), to inhibit its activation (antagonists) or to increase or decrease its response to an agonist (positive and negative allosteric modulators). Such other ligands include benzodiazepines (increase pore opening frequency; often the ingredient of sleep pills and anxiety medications), imidazopyridines (newer class of sleep medications), barbiturates (increase pore opening duration; used as sedatives), and certain steroids, called neuroactive steroids.

Among antagonists are picrotoxin (which blocks the channel pore) and bicuculline (which occupies the GABA site and prevents GABA from activating the receptor). The antagonist flumazenil is used medically to reverse the effects of the benzodiazepines.

A useful property of the many agonists and some antagonists is that they often have a greater interaction with GABA_A receptors which contain specific subunits. This allows one to determine which GABA_A receptor subunit combinations are prevalent in particular brain areas and provides a clue as to which subunit combinations may be responsible for behavioral effects of drugs acting at GABA_A receptors. Among the behavioral effects of such drugs are relief of anxiety (anxiolysis), muscle relaxation, sedation, anticonvulsion, and anesthesia.

See also

• GABA receptor
• GABA_B receptor
• GABA_C receptor
• Glycine receptor

References

• Chen K., Lia H.Z., Yea N., Zhanga J., and Wang J.J. 2005. Role of GABA_B receptors in GABA and baclofen-induced inhibition of adult rat cerebellar interpositus nucleus neurons in vitro. Brain Research Bulletin, 67(4), 310-318.
• Colquhoun D. and Sivilotti L.G. 2004. Function and structure in glycine receptors and some of their relatives. Trends in Neurosciences, 27(6), 337-344.
• Martin I.L., and Dunn S.M.J. 2002. "GABA Receptors". Tocris Cookson Ltd.
• Siegel G.J., Agranoff B.W., Fisher S.K., Albers R.W., and Uhler M.D. 1999. Basic Neurochemistry: Molecular, Cellular and Medical Aspects, Sixth Edition. GABA Receptor Physiology and Pharmacology. American Society for Neurochemistry. Lippincott Williams and Wilkins.
• Cossart R, Bernard C, Ben-Ari Y. 2005. Multiple facets of GABAergic neurons and synapses: multiple fates of GABA signalling in epilepsies. TRENDS in Neurosciences, 28(2), 108-115

External links

• Basic Neurochemistry: GABA Receptor Physiology and Pharmacology
• Dr. Dreyer's GABA-R webpage (University of Fribourg, Switzerland)

GABA ABC

GABA acts via three classes of receptors (GABA_A, GABA_B and GABA_C receptors) in the vertebrate CNS that differ in their structure, function, and pharmacology. The vast majority of fast GABA responses that are inhibited by bicuculline and picrotoxin and enchanced by benzodiazepines or barbiturates, result from the direct activation of an anion channel, that is referred to as the GABA_A receptor.

A second type of ionotropic GABA receptor (the GABA_C receptor) is insensitive to bicuculline, benzodiazepines and barbiturates, for review see Sieghart and Sperk, 2002. While GABA_A receptors are widely distributed in the CNS, the presence of GABA_C receptors is largely restricted to retinal bipolar or horizontal cells across vertebrate species. GABA_A receptors are heterooligomeric channel forming proteins, formed by different subunits that can belong to 8 distinct classes (α, β, γ, δ, ε, π, θ, and ρ)providing the potential for a huge number of receptor subtypes. In contrast, GABA_C receptors are homooligomeric receptors composed of ρ subunits. Although the GABA_C receptor terminology is still used frequently, these receptors are thought to be part of the GABA_A receptor family.

Fast responding GABA receptors are members of the Cys-loop ligand-gated ion channel superfamily (reviewed by Barnard et al., 1998) comprising nicotinic acetylcholine, GABA_A, GABA_C, glycine and 5-HT₃ receptors that possess a characteristic loop formed by a disulphide bond between two cysteine residues. GABA binding to the extracellular domain of GABA_A and GABA_C receptors triggers the opening of an intrinsic chloride-selective ionophore that drives the membrane potential towards the reversal potential for Cl¯ ions (about –80 mV) in neurons. As a consequence, the probability that action potentials will be generated by excitatory neurotransmission is decreased. But GABA_A receptor activation can also induce depolarizing responses, particularly in embryonic neurons. This phenomenon is due to increased intracellular Cl¯ concentration in certain neurons that occurs prior to the expression of the KCC2 transporter, which is principally responsible for Cl¯ extrusion. In adult brain, the GABA_A receptor can also mediate depolarizing responses under certain physiological and pathological conditions that involve intense receptor activation.

GABA_A Receptors

GABA_A Receptor Subunit Types

Initially two subunits of the GABA_A receptor named α and β were purified (Sigel et al., 1983, Sigel and Bernard, 1984). Subsequently the cDNAs coding for these subunits have been cloned (Schofield et al., 1987). So far 20 related GABA_A receptor subunits in mammals were identified 6α, 4β, 3γ, 1δ, 1ε, 1π, 1θ, and 3ρ (Barnard et al., 1998, Bonnert et al., 1999, Moragues et al., 2000). A mammalian counterpart of the avian γ₄ subunit (Harvey et al., 1993) has not yet been isolated by cDNA cloning and so is not included here. However, the β₄ subunit gene, likewise discovered in the chicken (Bateson et al., 1991), has more recently been shown to be present in humans (Levin et al., 1996). Sequence homology within same class of GABA_A receptor subunits is up to 80% and between subunit classes up to 40%. Homology with other members of Cys-loop receptor superfamily is about 10-20%. The subunits of this family are distributed in two groups, one containing the subunits forming anion selective receptors (GABA_A, GABA_C and glycine receptors), and one containing subunits forming cation selective receptors (5-HT³ and nicotinic receptors). For more complete insights in phylogenetical relationships within Cys-loop receptor family see reviews Ortells and Lunt, (1995) and Hervers and Luddens (1998). Splice variants add to the subunit diversity. Two forms of the γ₂ subunit are generated from one gene (Whiting et al., 1990, Kofuji et al., 1991). These receptors are differently expressed in the brain (Glencorse et al., 1992). Two forms are also known for both the β₂ and the β₄ subunits (Bateson et al., 1991, Harvey et al., 1994). In each case, the longer and shorter products were designated “L” and “S,” and differ by the presence of absence of short peptide in the long intracellular loop between TM3 and TM4. Splicing of exon-1 results in two alternative forms of the β³ subunit (Kirkness and Fraser, 1993). The α6 subunit is alternatively spliced in approximately 20% of its transcripts in rat brain, causing a deletion at the N-terminus of 10-amino acid residues (Korpi et al., 1994). Interestingly this deletion abolishes the functional receptor activity in all subunit combinations tested so far.

Regional Distribution of GABA_A Receptor Subunits in the Brain

Subunit isoform distribution in brain has been studied by using “in situ” hybridization at the mRNA level (Laurie et al., 1992, Persohn et al., 1992, Wisden and Seeburg, 1992, Miralles et al., 1994, Bonnert et al., 1999, Sinkkonen et al., 2000) and by using immuno-histochemical studies at the protein level (Benke et al., 1991a, b, c, Zimprich, et al., 1991, Fritschy et al., 1992, Gutierrez et al., 1994, Fritschy et al., 1995, Sperk et al., 1997, Kultas-Ilinsky et al., 1998, Fritschy et al., 1998, Moragues et al., 2000, Pirker et al., 2000, Schwarzer et al., 2001, Moragues et al., 2002, Moragues et al., 2003, Plotl et al., 2003). The individual subunits exhibit a distinct but overlapping regional and cellular distribution. Subunits α₁, β₁, β₂, β₃ and γ₂ are found throughout the brain, although differences in their distribution were observed.

The α₁ subunit is the most abundant, subunits α₂, α₃, α₄, α₅, α₆, γ₁, and δ are more confined to certain brain areas. The α2 subunits are preferentially located in forebrain areas. The highest concentrations were found in olfactory bulb, striatum, nucleus accumbens, septum, dentate gyros, amygdale and hypothalamus. α₂ subunits were less abundant in thalamus (except reticular nucleus), midbrain and brainstem areas. α³ subunits were strongly expressed the glomerular and external plexiform layers of the olfactory bulb, in the inner layers of the cerebral cortex, the reticular thalamic nucleus, and the zonal and superficial layers of the superior colliculus, the amygdala and cranial nerve nuclei. Subunit α₄ was strongly expressed in the thalamus, dentate gyros, olfactory tubercle and basal ganglia (Benke et al., 1997). The α₅ subunit immunoreactivity was strongest in Ammon’s horn, the olfactory bulb and hypothalamus, whereas the α₆ subunit is exclusively expressed in granule cells of the cerebellum and the cochlear nucleus (Pirker et al., 2000). The β subunits are widely distributed. The β₂ subunit is one of the most widely distributed subunits in the brain. β₁ and β₃ subunits are less abundant (Benke et al., 1994).

Among the γ subunits the γ₂ is most widely distributed throughout the brain, whereas γ₁ and γ₃ are relatively rare (Somogyi et al., 1996). The subunit γ₁ is the rarest subunit and exhibits a quite specific distribution in the brain. It is preferentially located in the central and medial amygdaloid nuclei, in pallidal areas, the substantia nigra pars reticulate and the inferior olive. In contrast, the γ³ subunit is expressed in most brain areas but with low abundancy. The δ subunit can be co-localized with the α₄ subunit, e.g. in the thalamus, striatum outer layers of the cortex and in the dentate molecular layer and in the neonatal hippocampus (Sur et al., 1999, Bencsits et al., 1999, Didelon et al., 2000). In cerebellum it is co-distributed with α₆ subunit (Pirker et al., 2000). The π subunit was detected in several peripheral human tissues as well as in the brain (hippocampus and temporal cortex) and was particularly abundant in the uterus (Hedblom and Kirkness, 1997) So far no study investigating the detailed regional distribution of the π subunit has been published. The θ subunit (Bonnert et al., 1999) seems to be expressed in various regions, including the hypothalamus, amygdala, hippocampus, substantia nirga, dorsal raphe and locus coeruleus (Sinkkonen et al., 2000, Moragues et al., 2002). θ subunits showed strikingly overlapping expression patterns with ε subunits throughout the brain, especially in the septum, preoptic areas, various hypothalamic nuclei, amygdala, and thalamus, as well as in monoaminergic groups (Moragues et al., 2002). As with the ε subunit, there were some discrepancies in the cDNA sequence obtained by different groups (Bonnert et al., 1999, Sinkkonen et al., 2000).

The ρ subunits seem to be preferentially expressed in the retina. Immunohistochemistry in the retina using an antibody recognizing all 3 ρ subunits revealed a staining pattern restricted to the terminals of bipolar cells in the inner plexiform layer which did not overlap with GABA_A α or β subunits (Enz et al., 1996, Koulen et al., 1998, Fletcher et al., 1998, Koulen, 1999). mRNA encoding ρ subunits, however, is present also in the superior colliculus, dorsal lateral geniculate nucleus and cerebral Purkinje sells (Boue-Grabot et al., 1998, Wegelius et al., 1998). In addition, bicuculline-resistant and baclofen-independent GABA effects were reported in the cerebellum (Drew and Johnston, 1984, Drew and Johnston, 1992), superior colliculus (Arakawa et al., 1988, Clark et al., 2001), amygdala (Delaney and Sah, 1999), hippocampus (Martina et al., 1996, Cherubini et al., 1998, Didelon et al., 2002), dorsal geniculate cells (Zhu and Lo, 1999) and spinal cord (Park et al., 1999). This indicates that ρ subunits may be present in many CNS regions and are more prevalent than previously suspected.

Architecture of Recombinant GABA_A Receptors

Architecture of Recombinant GABA_A Receptor Subunits

All GABA_A receptor subunits are composed of a large N-terminal extracellular domain, four transmembrane (TM) domains, and a large intracellular loop between TM3 and TM4 (Schofield et al., 1987). Subunits are up to 460 amino acids in length. The N-terminal extracellular domain is carrying several potential sites for N-linked glycosylation (Buller et al., 1994) and two conserved cysteine residues. Upon receptor assembly homologous parts of this domain form at subunit interfaces binding sites for agonists and ligands of the benzodiazepine binding site. Other binding sites as those for anesthetics, barbiturates, ethanol, furosemide, zinc and some other compounds located within transmembrane domains. Each subunit contributes to the channel lining that is largely formed by residues of TM2, and possibly of TM3 membrane-spanning segments (Xu and Akabas, 1996, Williams and Akabas, 1999, Goren et al., 2004).

The three-dimensional structure of a related receptor – the nicotinic acetylcholine receptor (Unwin, 1993, Miyazawa et al., 1999, Miyazawa et al., 2003) indicates that the transmembrane domains have an α-helical structure, the pore is shaped by an inner ring of α-helices, which curve radially to create a tapering path for the ions, and an outer ring of α-helices, which coil around each other and shield the inner ring from the lipids. The gate is a constricting hydrophobic girdle at the middle of the lipid bilayer, formed by weak interactions between neighboring inner helices.

How Many Subunits Make a GABA_A Receptor?

As mentioned above the GABA_A receptor subunits share amino acid sequence homology with the subunits of the nicotinic acetylcholine receptors. The muscle type of the nicotinic acetylcholine (nAChR) receptor occurs in Torpedo electric organ in such a high density that it is possible to prepare membranes containing a lattice of the receptors, from which a low-resolution three-dimensional structure of the molecule could be obtained by electron optical diffraction techniques (Toyoshima and Unwin, 1988, Unwin, 1993, Miyazawa et al., 2003, Unwin, 2003). Those studies clearly showed that the muscle type nicotinic receptor is pentameric, with the ion channel located in the center of a rosette. For the GABA_A receptors, the situation is more complex as no such rich sources exist. Using purified GABA_A receptors from pig brain cortex combined with image analysis in the electron microscope, dispersed single receptor molecules have been visualized and analyzed. This method indicated a pentamer (Nayeem et al., 1994). Furthermore, the negatively stained images indicated a central pore in the pentameric rosette.

Independent evidence to support a pentameric structure has been obtained in several ways. Hydrodynamic estimates of the size of GABA_A receptors in solution, either native (Mamalaki et al., 1989) or α₁β³γ₂ recombinant receptors (Tretter et al., 1997) are consistent with the molecular weight of a pentamer. Further, the integral ratios of the subunits combined in several forms of functional recombinant receptors, as determined by diverse methods, fit best in each case with total of five subunits (Backus et al., 1993, Im et al., 1995, Chang et al., 1996, Tretter et al., 1997, Ferrar et al., 1999). A powerful way to gain insight into the arrangement of subunits in GABA_A receptors and their stoichiometry is the use of a predefined alignment of subunits by producing linked subunit constructs with the aid of gene fusion (Im et al., 1995, Baumann et al., 2001, Baumann et al., 2002). Analysis of receptors formed by linked subunits also indicated a pentameric stoichiometry.

Assembly of GABA_A Receptor Subunits

GABA_A receptor assembly occurs within the endoplasmic reticulum (ER) (Czajkowski and Farb, 1989, Kittler et al., 2000, Moss and Smart, 2001, Kittler et al., 2002). Their distinct subunit compositions may provide distinct functional properties e.g. modulation by endogenous ligands such as neurosteroids (Twyman and Macdonald, 1992, Wohlfarth et al., 2002, Akk and Steinbach, 2003, Bianchi and Macdonald, 2003) or second messenger systems (Angelotti et al., 1993, Moss and Smart, 2001), subcellular localization (Connolly et al., 1996a), or long term differences in the regulation of types of receptor surface expression (Connolly et al., 1999a,b). Many neuron express multiple receptor subunit mRNAs simultaneously (Wisden and Seeburg, 1992, Sieghart and Sperk, 2002), suggesting that cellular mechanisms for differential receptor assembly may also exist. To achieve the correct arrangement of subunits around the pore, each subunit must form specific contacts, assembly signals, on interfaces contacting with neighbors subunits. The presence of such multiple assembly signals is capable of differential interaction with other subunits may permit construction of different GABA_A receptors. Individual subunits may not be committed to a particular receptor subtype, but may function as universal building blocks in the generation of diverse receptor compositions.

In the α1 subunit residues (54–68) on (-) side were identified as important for assembly with β subunits (Taylor et al., 2000, Klausberger et al., 2000, Sarto et al., 2002a). An additional interaction site included the residues (80–100) on α₁ subunit (+) interface, which are believed to be important for assembly with the γ₂ subunit (Klausberger et al., 2001). Subsequently, single amino acid residues implicated in assembly were identified. Thus it was shown that a single amino acid residue Q67 (Taylor et al., 2000) is important for assembly of α₁ with β³ but not with γ₂ subunits. Conversion of a single amino acid in α1 to that of γ2 (R66A) was shown to be sufficient to alter the assembly profile of the α₁ subunit to that of the γ₂ subunit. It was also shown that presence of this residue is required for the assembly of α₁β₂ but not α₁β₁ or α₁β₃ (Bollan et al., 2003a,b). Two tryptophan residues α₁W69 and α₂W94, on the rat α1 subunit were found to be critical for the assembly of the GABA_A receptor pentamer (Srinivasan et al., 1999).

In β₂ and β₃ subunits it was found that region (52–66) on (-) interface (Taylor et al., 1999, Klausberger et al., 2000, Sarto et al., 2002a) is important for assembly with α₁ subunits. An additional region (76–89) located on β₃ subunit (+) interface is important for assembly with α1 subunit was identified later (Ehya et al., 2003). In the γ subunits amino acid sequences γ₂ (67–81) (Sarto et al., 2002a), γ₃ (70–84) (Sarto et al., 2002b) located at (-) interface and γ2(83–90) and γ₂ (91–104) located at (+) interface (Klausberger et al., 2000) were identified as sites important for assembly with α₁ and β³ subunits. There is also a report that a singe amino acid γ₂W82 residing on (-) interface upon mutation to cysteine failed to express with α₁ and β₂ subunits (Teissere and Czajkowski, 2001). Regions α₁ (54–68), β₂ (52–66), and γ₂ (67–81), are located in homologous regions of (-) sides of the different subunits (Sarto et al., 2002b). It has been reported that dimer or trimer assembly intermediates of GABA_A receptor subunits can form binding sites for [³H]muscimol and [³H]Ro15-1788 (Klausberger et al., 2001).

Interestingly, these assembly signals or intersubunit contact points at the α, β and γ subunits (Taylor et al., 2000, Klausberger et al., 2000, Klausberger et al., 2001, Sarto et al., 2002a,b) overlap with the GABA (Boileau et al., 1999b) and benzodiazepine binding sites (Buhr et al., 1997, Boileau et al., 1999a, Teissere et al., 2001, Sigel, 2002) formed at subunit interfaces between the α/β and α/γ subunits.

Subunit Composition of Recombinant GABA_A Receptors

With the application of molecular biology approaches, in the late 1980s and 1990s, it soon became clear that a family of GABA_A receptor subtypes composed from different subunits exists within the brain. If all these subunits could randomly co-assemble with each other, more than 151,887 GABA_A receptors subtypes with distinct subunit composition, arrangement would be formed (Burt and Kamatchi, 1991). Not all subunits can assemble efficiently with each other and form functional receptors.

Homo-oligomeric Recombinant GABA_A Receptors

Recombinant expression studies have indicated that at least some of the GABA_A receptor subunits can form homo-oligomers. The extent of formation of these homo-oligomers, however, varies dramatically. Whereas some are robustly formed in all recombinant expression systems, others are formed with low efficiency only (Blair et al., 1988). Xenopus oocytes or HEK-293 cells have been used mainly as host cells.

A robust expression of GABA-activated homo-oligomeric chloride channels was observed with ρ subunits (Cutting et al., 1991, Shimada et al., 1992, Kusama et al., 1993a, b, Wang et al., 1994, Shingai et al., 1996). To smaller extent expression of homo-oligomeric receptors was observed with β₁ or β³ subunits (Connolly et al., 1996a, b, Krishek et al., 1996b, Wooltorton et al., 1997b) and γ_2L subunits (Martinez-Torres and Miledi, 2004).

Interestingly, channels formed by murine or rat β₁ (Sigel et al., 1989, Krishek et al., 1996b) or β³ subunits (Wooltorton et al., 1997b) were open in the absence of GABA, but could be inhibited with channel blocker picrotoxin. This effect seems to be species dependent, because human or bovine β₁ subunits seem to be able to form homo-oligomeric channels closed in the absence of GABA (Pritchett et al., 1988, Krishek et al., 1996b, Sanna et al., 1995).

Hetero-oligomeric Recombinant GABA_A Receptors Composed of Two Different Subunits

The efficiency of receptor formation of two different subunits depends on the subunit combination. Whereas different αβ subunit combinations are expressed efficiently and form GABA-activated channels in all systems investigated, conflicting results were obtained with αγ or βγ subunit combinations (Verdoorn et al., 1990, Sigel et al., 1990, Knoflach et al., 1992, Angelotti et al., 1993). The efficiency of formation of pentameric α₁γ₂ or β³γ₂ receptors heterologously expressed in HEK-293 cells seems to be low (Tretter et al., 1997). For the cells co-expressing β³ and γ_2L subunits, γ_2L could be detected on the surface of only about 15% of cells, indicating that most of the receptors formed in these cells were homo-oligomeric β³ receptors (Taylor et al., 1999, Bollan et al., 2003a, b). β₁γ_2S, β₂γ_2Sand β³γ_2S receptors also formed in HEK-293 cells to a comparable extent and exhibit pharmacological properties distinct from that of homo-oligomeric β_1-3 receptor (Taylor et al., 1999, Hamon et al., 2003). It has been observed that α₁γ₂ or β₂γ_2L subunits combinations were retained within the endoplasmatic reticulum (Connolly et al., 1996a, b). It is thus possible that receptors composed of these subunit combinations can only be formed under certain experimental conditions, such as in the presence of suitable chaperons, at high subunit concentrations due to high synthesis rates (conditions that are present in some recombinant receptor systems). No information is available on the possible formation of GABA_A receptors composed of αδ, βδ or γδ subunits. No functional channels, however, were formed on co-transfection of α₁ε or β₁ε (Whiting et al., 1997) or of α₁π or β₁π (Hedblom et al., 1997) subunit combinations.

In contrast, different ρ subunits can combine with each other and might also co-assemble to functional receptors in vivo (Enz and Cutting, 1998). Although in one study it was demonstrated that ρ subunits are unable to assemble with α₁, β₁ or γ₂ subunits (Enz and Cutting, 1998), other studies indicated that ρ subunits can assemble with γ₂ subunits and possibly also with glycine receptor subunits, and also form functional receptors found in certain cell types of the retina (Pan et al., 1997, Qian and Ripps, 1999, Pan et al., 2000).

Hetero-oligomeric Recombinant GABA_A Receptors Composed of Three and More Different Subunits

Recombinant receptors subtypes composed of an α, a β and a γ subunit mainly have been studied so far (Sieghart, 1995, Herves and Luddens, 1998, Sieghart and Sperk, 2002). Assuming the stoichiometry of 2:2:1 these receptors can have at least one of three general compositions: 2α/2β/γ; 2α/β/2γ; α/2β/2γ. Here, a notation is introduced in which the numeral represents the number of molecules of a given subunit class (α, β, etc.) present in one receptor molecule and not the isoform identity within that class. Based on abundance of co-expression, it is assumed that α₁β₂γ₂ represents the most abundant GABA_A receptor in adult mammalian brain (Herves and Luddens, 1998, Sieghart and Sperk, 2002). Additional cases such as 3α/β/γ, α/3β/γ and α/β/3γ are theoretically possible, but immunoprecipitation (Tretter et al., 1997), measurements of electrophysiological properties (Backus et al., 1993, Chang et al., 1996) and fluorescence energy transfer (Ferrar et al., 1999) have excluded (at least in those cases) the presence of three identical subunit isoforms in one receptor molecule.

Additional studies have indicated that receptors containing two different α subunit isoforms, in combination with a β and a γ subunit can assemble and exhibit properties that are distinct from those of receptors containing only a single type of α subunit (Verdoorn et al., 1990, Sigel et al., 1990, Polenzani et al., 1991, Verdoorn, 1994, Sigel and Baur, 2000, Hansen et al., 2001).

Similarly, it has been demonstrated that receptors containing two different types of β subunits together with one of α and γ subunit are able to assemble and to exhibit properties different form receptors that contain only a single β subunit subtype (Fisher and Macdonald, 1997). Finally, it has been demonstrated that recombinant receptors composed of α₁, β₁, the long splice variant of γ_2L, and δ (α₁β₂γ_2Lδ) or α₁, β₃, γ₃, and π (α₁β₃π and α₁β₃γ₃π) subunits can also be formed and exhibit properties distinct from those of α₁β₁γ_2L or α₁β₁δ receptors (Saxena and MacDonald, 1994, Hansen et al., 2001, Hevers et al., 2000) or from those of α₁β³ and α₁β₃γ₃ receptors (Neelands and Macdonald, 1999) respectively. Although experiments investigating the expression of five different subunits have been performed in Xenopus oocytes, the results obtained were difficult to interpret (Sigel et al., 1990). This is not surprising because from the five different subunits simultaneously expressed in the oocytes a variety of different receptor subtypes composed of 3, 4, or 5 different subunits could have been formed, that all could have contributed to the chloride current measured in these cells. This problem could be solved by linking multi-subunits by gene fusion. This methodology has already been applied successfully to GABA_A receptors (Baumann et al., 2001, Baumann et al., 2002, Baumann et al., 2003).

Functional Architecture of GABA_A Receptors

The N-terminal domains of GABA_A receptor subunits are implicated in receptor assembly and in formation of agonist and benzodiazepine binding sites. Two agonist binding sites are harbored by α(-)/β(+) subunit interfaces (Boileau et al., 1999b, Teissere and Czajkowski, 2001). It was found recently that even though agonist sites are located at similar interfaces formed by identical (-) sides of α and (+) sides of β subunits, they have dissimilar properties: site 2 has an approximately threefold higher affinity for GABA than site 1, whereas muscimol and bicuculline show some preference for site 1 (Baumann et al., 2003). The benzodiazepine binding site is located at α(+) and γ(-) subunit interface. Interestingly, domains involved in the formation of the GABA and benzodiazepine binding sites are homologous (reviewed in Sigel, 2002). A large body of evidence has been collected suggesting that anesthetics, barbiturates, alcohols and number of other drugs share some overlapping structural determinants for their actions on the GABA_A receptor. All these allosteric sites are located within the gating domain and it was observed that compounds acting at these sites were capable of direct activation of channel in the absence of GABA (Belelli et al., 1999, Akk and Steinbach, 2000).

On the cytoplasmatic side at the entry to channel pore is located binding site for channel blockers. This chemically inhomogeneous group of compounds is comprised from substances acting as physical plugs. Prototypic compounds of this class are picrotoxinin (Inoue and Akaike, 1988, Yoon et al., 1993), the bicyclic caged compound TBPS (Squires et al., 1983, Supavilai and Karobath, 1983). Properties of this binding site were found to be similar among different αβγ receptors (Bell-Horner et al., 2000), however, some unusual subunit combinations, like receptors formed by β subunits (Sigel et al., 1989, Krishek et al., 1996b, Wooltorton et al., 1997b) showed increased sensitivity to channel blockers. In following the binding sites for the receptor agonist GABA, the benzodiazepine binding pocket, binding sites located within transmembrane domains of subunits and binding site of channel blockers are discussed in more detail.

Agonist Binding Sites of the GABA_A Receptor

Ligands Acting at Agonist Sites of GABA_A Receptors

The endogenous activator of GABA_A receptors is GABA. Various compounds of different type and intrinsic activity are also recognized by the agonist binding site of the GABA_A receptor. Binding of agonist is coupled to the opening of the channel, the so-called channel gating. Partial agonists differ from agonists in respect of channel opening efficacy. Binding of a competitive antagonist is stabilizing the closed state of the receptor channel. Competitive antagonists are viewed as classic competitive inhibitors of GABA_A receptor (Macdonald and Olsen, 1994), but there are indications that they can induce conformational changes (Ueno et al., 1997, Bianchi and Macdonald, 2001, Wagner and Czajkowski, 2001).

Architecture of the Agonist Binding Sites

Residues implicated in agonist binding are assigned to at least six different non-contiguous extracellular N-terminal regions of the α and β subunits. These regions have been designated loops A–F in the homologous nicotinic acetylcholine receptor (Corringer et al., 2000, Le Novere et al., 2002a). In GABA_A receptors, the agonist binding site is formed by (-) side of α and (+) side of β subunit. Residues in different loops likely have different functional roles. Some residues may directly contact ligand, some may be important for maintaining the structural integrity of the binding site, and others may mediate local conformational movements within the site.

The following residues are thought to take part in the formation of the agonist site. On the α₁ subunit, residues identified include F64 (Sigel et al., 1992, Smith and Olsen, 1994), R66, S68 (Boileau et al., 1999) (on loop D, K116, R119, and I120 (Westh-Hansen et al., 1997, Westh-Hansen et al., 1999, Hartvig et al., 2000) (on loop E) and V178, V180, D183 (Newell and Czajkowski, 2003) (on loop F). Complementary residues in the β₂ subunit include Y97, L99 (Boileau et al., 2002) (on loop A), Y157, T160 (Amin and Weiss, 1993) (on loop B), T202, S204, Y205, R207, and S209 (Amin and Weiss, 1993, Wagner and Czajkowski, 2001) (on loop C) have been identified.

The spatial arrangement of these residues was unclear until direct crystallographic evidence was obtained on protein involved in synaptic transmission of the snail Lymnaea stagnalis has helped to visualize residues forming the binding site after homology modeling. This water-soluble protein is called acetylcholine binding protein (AChBP). AChBP subunit is 210 residues long, forms a stable homopentamer (Brejc et al., 2001, Smit et al., 2003) and shares 24% sequence homology with the N-terminal part of human α₇ nicotinic receptor subunit and about 15% with subunits of GABA_A receptor family.

The Benzodiazepine Binding Site

Ligands of the Benzodiazepine Binding Site

In 1957, scientists at a drug company (Hoffmann-La Roche) by accident discovered that a new compound, chlordiazepoxide reduced fear in animals. This compound was a benzodiazepine and its discovery ushered a new era in treatment of anxiety and related disorders. Since then, the number of compounds with the structure of benzodiazepine template reached more than 3000. Among the pharmacological agents that allosterically modulate GABA_A receptors, the benzodiazepines have gained major clinical relevance (Mohler et al., 1996a,b, Sieghart, 2003). Present evidence suggests that GABA_A receptors are the only effector sites of benzodiazepines in the central nervous system. Ligands of the benzodiazepine binding site have been subdivided into three classes according to their intrinsic activity: positive allosteric modulators, negative allosteric modulatorsand antagonists. The names “agonist”, “inverse agonist” and “antagonist” are also used for these compounds. Classical benzodiazepines upon binding to GABA_A receptor exert their positive allosteric effect by increasing the affinity of GABA for its binding sites without affecting maximum response. This results in increased probability of channel opening (Rogers et al., 1994). Antagonists of the benzodiazepine site do not affect GABA-elicited responses. However, they prevent positive allosteric modulators from binding and thus, from allosteric modulation of receptor function. Negative allosteric modulators have opposite effects to positive allosteric modulators, decreasing affinity for GABA. An additional, “peripheral” recognition site of benzodiazepines, structurally and functionally unrelated to GABA_A receptors, is located at 18 kDa protein found in the mitochondrial membrane (for review see Papadopoulos et al., 2001).

Architecture of the Benzodiazepine Binding Site in Recombinant αβγ Receptors

The benzodiazepine binding site has been shown to be located at the interface between the α- and γ-subunits, with residues from each subunit contributing to the binding site (Casalotti et al., 1986, Deng et al., 1986, Pritchett et al., 1989, Smith and Olsen, 1995, Sigel and Buhr, 1997, Sigel, 2002). Photoaffinity labeling of the receptor by benzodiazepines [³H]flunitrazepam and [³H]Ro15–4513 has been performed (Mohler et al., 1980, Fuchs et al., 1988, Stephenson et al., 1990, McKernan et al., 1995, Davies et al., 1996, Smith and Olsen, 2000, Sawyer et al., 2002). The residues H101 (rat numbering) (McKernan et al., 1995, Duncalfe and Dunn, 1996, Duncalfe et al., 1996, Smith and Olsen, 2000) and P97 (Smith and Olsen, 2000) have been shown to be the major sites of incorporation of [³H]Flunitrazepam into the α₁ subunits. [³H]Ro15-4513 can also be photoincorporated into α subunits of the GABA_A receptor (Sieghart et al., 1987). The amino acid(s) photolabeled by [³H]Ro15-4513 are contained within a subunit fragment extending from residue 104 to the C terminus of the α₁ subunit (Duncalfe and Dunn, 1996), possibly within amino acids 247–289 spanning the end of the TM1 to the beginning of the TM3 (Davies and Dunn, 1998). The results from a recent study suggest that [³H]Ro15-4513 is photoincorporated into α₁Y209 and in homologous positions in the α₂ and α³ subunits (Sawyer et al., 2002).

Extensive mutagenesis experiments have also identified other α₁ residues implicated in benzodiazepine binding. The significance of α₁H101 has initially been demonstrated in studies in which this residue has been substituted with arginine, the native residue at the homologous position in α4 and α6 subunits (Wieland et al., 1992). Substitution of this histidine by arginine resulted in about 500-800 fold decrease in affinity of classical benzodiazepines (Wieland et al., 1992). Extensive mutational analysis of α₁H101 residue has also revealed its implication in allosteric coupling between GABA and benzodiazepine binding sites (Davies et al., 1998b).

The following residues in the α₁ subunit were shown to either affect benzodiazepine sensitivity in functional assays or benzodiazepine affinity in binding studies, Y159 (Amin et al., 1997) and Y209 (Amin et al., 1997, Buhr et al., 1997b), T162 (Wieland and Luddens, 1994), G200 (Pritchett and Seeburg, 1991, Schaerer et al., 1998, Wingrove et al., 2002), T206 (Buhr et al., 1997b), V211 (Casula et al., 2001) and I215 (Strakhova et al., 2000). In the γ2 subunit M57 (Buhr and Sigel, 1997) and Y59 (Kucken et al., 2000) were found to be essential determinants for conferring high-affinity for classical and atypical benzodiazepines. The F77 residue was absolutely crucial for maintaining ability of benzodiazepine binding site to recognize its classical ligands (Buhr et al., 1997a, Wingrove et al., 1997, Sigel et al., 1998). It should be noted that this residue is homologous to F64 in α subunit, which has been previously shown to be a key determinant of the GABA binding site, initially suggesting a conservation of motifs between different ligand binding sites on the GABA_A receptor (Sigel et al., 1992, Smith and Olsen, 1994, Buhr et al., 1997a, Sigel et al., 1998). Residue M130 is required for high affinity binding of benzodiazepine binding site ligands (Buhr and Sigel, 1997, Wingrove et al., 1997, Sigel et al., 1998). And finally, threonine residue at position 142 was implicated in the efficacy of benzodiazepine binding site ligands (Mihic et al., 1994). Recently using analogy to a GABA binding pocket residues A79 and T81 which are clustered on a β-strand around F77 were found to line up the part of binding pocket (Kucken et al., 2000, Teissere and Czajkowski, 2001).

Benzodiazepine Binding Sites May Be Composed of Different α and γ Subunit Isoforms

Different α and γ subunit isoforms can assemble to form a benzodiazepine binding site thereby imposing different pharmacological properties. Benzodiazepine receptors have been classified pharmacologically into those which recognize the classical, 5-phenyl-1,4-benzodiazepines (for example diazepam and flunitrazepam) referred to as ‘diazepam-sensitive’ receptor and those which do not recognize these ligands referred to as ‘diazepam-insensitive’ receptor (Malminiemi and Korpi, 1989, Hadingham et al., 1996, Knoflach et al., 1996). As described above, the residue at position 101 of α₁ (and homologous positions in other α subunits) has been shown to determine the affinity for diazepam. α₁, α₂, α³ or α₅ have a histidine in this position and display high affinity for diazepam, while α₄ or α₆ have arginine at the homologous position do not bind diazepam (Wieland et al., 1992, Dunn et al., 1999, Kelly et al., 2002). α₁, α₂, α³ and α₅ subunit containing receptors can be further subdivided by their affinity to CL218872 with the higher affinity α₁ subunit containing receptors have a higher affinity and are referred to BZI type receptors and the α₂, α₂ or α₅ subunit containing receptors to BZII type receptors (Pritchett and Seeburg, 1991, Yang et al., 1995). Selectivity for CL218872 and zolpidem of α₁ subunits over α₆ is conferred mainly by α₆T161 (Wieland and Luddens, 1994, Renard et al., 1999), α₁G201 (Pritchett and Seeburg, 1991, Schaerer et al., 1998), α₁S205 (Wieland and Luddens, 1994, Renard et al., 1999) and α₁V211 (Casula et al., 2001).

It is noticeable that the subunits conferring the higher affinity to zolpidem have the smaller amino acid residues at both positions 201 and 211, suggesting a steric role for these residues in benzodiazepine selectivity. It was proposed that a similar mechanism also underlies more than 1000-fold decrease in affinity for diazepam, flunitrazepam, zolpidem and CL218872 at α₆-containing receptors compared to those having α1 (Luddens et al., 1990, Hadingham et al., 1996, Casula et al., 2001, Wingrove et al., 2002). Another residue conferring insensitivity of α6 subunit containing receptors to β-carboline β-CCE is α6N204. Introduction of a point mutation α₆N204S or α₆N204I, found in homologous position of α₁ or α₄ subunits restored affinity (Derry et al., 2004).

From two studies of Stephenson et al. (1990), Puia et al. (1991) and McKernan et al. (1995) it was clear that the type of γ subunit also contributes significantly to the properties of the benzodiazepine binding site. GABA_A receptors containing a γ₁ subunit have a >5000-fold lower affinity for the antagonist Ro15-1788 than do those containing a γ₂ or γ³ subunit, whereas γ₁- and γ³-containing receptors have a about 100-fold reduced affinity for zolpidem and 10–30-fold lower affinity for flunitrazepam than do receptors containing γ₂ (Hadingham et al., 1995, Benke et al., 1996, Wingrove et al., 1997). Two amino acid residues are determining selectivity of zolpidem for γ₂ subunit containing receptors (Wingrove et al., 1997, Buhr et al., 1997a, Buhr and Sigel, 1997). These are γ₂F77 and γ₂M130; γ₁ subunit has an isoleucine (γ₁I79) at position homologous to γ₂F77 and additionally γ₁ and γ³ subunits contain a leucine in position homologous to γ₂M130 (γ₁L132 and γ³L133). Introduction of point mutations γ₁I79F, γ₁L132M in γ₁ subunit and γ³L133M in γ³ restored zolpidem affinity (Wingrove et al., 1997, Buhr and Sigel, 1997).

Relative Position of Ligands in the Benzodiazepine Binding Site

Many attempts have been made to characterize interactions of benzodiazepine binding site ligands in their binding pocket and to superimpose positive and negative allosteric modulators and antagonists (Borea et al., 1987, Villar et al., 1989, Schove et al., 1994, Zhang et al., 1995b, Huang et al., 1998, Huang et al., 1999, He et al., 2000, Marder et al., 2001, Verli et al., 2002). All these studies have investigated quantitative-structure affinity/activity relationships using chemically related compounds and have inferred type of interacting points e.g. lipophilic, aromatic, H-donor, H-acceptor and distances between these interaction centers. Such studies, have been found to be useful to estimate binding affinities and mode of action of newly designed ligands of the benzodiazepine binding site, however have very limited use for positioning of a ligand in the binding pocket. As detailed above, a soluble remote homologue of the N-terminal extracellular domain of nicotinic acetylcholine receptors (nAChR), the acetylcholine binding protein (AChBP), has been recently crystallized (Brejc et al., 2001). This crystal structure, featuring a novel fold of modified immunoglobulin-like topology, was used to construct homology models of the N-terminal domain of other superfamily members. A few models of nAChRs (Fruchart-Gaillard et al., 2002, Le Novere et al., 2002b, Schapira et al., 2002) and, also of GABA_A receptors (Cromer et al., 2002, Trudell, 2002, Ernst et al., 2003) have been published.

These models of the extracellular domain of GABA_A receptors provide a tool for the visualization of existing data (Holden and Czajkowski, 2002, Kash et al., 2003, Kucken et al., 2003a, Kash et al., 2004) and planning of rational mutagenesis studies. However, authors indicate many uncertainties in these models (Ernst et al., 2003). Their limited accuracy results from the low sequence homology between GABA_A receptor subunits and AChBP template. Computational docking in models of the benzodiazepine site is presently hampered (Ernst et al., 2003). In spite of these problems possible positioning of the imidazobenzodiazepine Ro15-4513 has been suggested (Sawyer et al., 2002). [³H]Flunitrazepam primarily labeled residue H101 (rat numbering) (McKernan et al., 1995, Duncalfe et al., 1996, Smith and Olsen, 2000) and P97 (Smith and Olsen, 2000) and it has been assumed that in the binding pocket flunitrazepam molecule is pointed in the direction of these amino acids (reactive group in this case presumably not diffusing away from the primary site of radioactivity incorporation). In case of [³H]Ro15-4513 was shown to label α₁Y209 and homologous positions of the α₂ and α³ subunits (Sawyer et al., 2002). It is, however, difficult to infer the precise geometrical orientation from these labeling studies.

Observations relevant for the interaction of ligands of the benzodiazepine binding site with the γ subunit were made in two different studies. In the first affinities of ligands based on two templates – imidazobenzodiazepines (Ro15-1788-like) and 5-phenyl-1,4-benzodiazepine (diazepam- and flunitrazepam-like) to wild-type and mutant receptors were determined (Sigel et al., 1998). The authors concluded, that the extra hydroxyl group in tyrosine introduced in the mutant γ₂F77Y interferes with the phenyl moiety of benzodiazepine and therefore γ₂F77 should be close to the phenyl substituent in 5-phenyl-1,4-benzodiazepines (Sigel et al., 1998). Another study, where the size of moiety occupying the 3’-imidazo substituent (ester group in Ro15-1788/Ro15-4513) was varied together with the volume of the residue introduced in the γ₂A79 position suggested that that Ro 15-4513 spans the binding site between α₁Y209 and γ₂A79, with the azide substituent facing the α₁ subunit and the 3’-imidazo substituent facing the γ₂ subunit. Computational docking of Ro 15-4513 and Ro15-1788 into the benzodiazepine binding site performed in the same study position the 3’-imidazo substituent near γ₂A79 and γ₂T81 residues (Kucken et al., 2003).

Allosteric Sites within Transmembrane Domains

Compounds Acting at Allosteric Sites within Transmembrane Domains

Modulation of GABA_A receptor function by most volatile, intravenous, general anesthetics (Belelli et al., 1997, Belelli et al., 1999, Krasowski et al., 2001a,b, Korpi et al., 2002, Siegwart et al., 2002, Bali and Akabas, 2004), alcohols (Wick et al., 1998, Ueno et al., 1999, Mascia et al., 2000, Ueno et al., 2000), anticonvulsants (Vaught, and Wauquier, 1991, Wafford et al., 1994) and allosteric antagonists (Korpi et al., 1995, Thompson et al., 1999a, Fisher, 2002) is mediated via allosteric binding sites located within transmembrane domains of α and β subunits. At high concentration some compounds like propofol, barbiturates, loreclezole and etomidate can directly gate ion channel of the GABA_A receptor in the absence of its agonist, GABA (Sanna et al., 1996, Akk and Steinbach, 2000, Steinbach and Akk, 2001). At low concentrations they modulate GABA-induced openings (Belelli et al., 1999), and, depending on the type of compound, this potentiation of GABA-gated currents appears to alter receptor deactivation and/or desensitization (Mozrzymas et al., 1999, Li and Pearce, 2000, Bai et al., 2001).

Allosteric Binding Sites Located within α and β Subunits

A set of residues located in the transmembrane domains 1-4 of GABA_A receptor α and β subunits confer potency of various clinically used compounds. Residues implicated in formation of these binding sites are located within homologous domains of α and β subunits. It is worth noting, that the same transmembrane regions have been described as an integral part of the channel gating domain of the GABA_A receptor (Xu and Akabas, 1996, Horenstein et al., 2001) and other ligand-gated ion channels (Le Novere et al., 2002a, Unwin, 2003, Miyazawa et al., 2003).

Residue α₁G223F of TM1 segment of α subunits affects receptor gating induced by pentobarbital and propofol (Engblom et al., 2002), another residue α₂L232F was implicated in halothane action (Jenkins et al., 2001). It was found that a single amino acid in α₆ subunit, α₆Ι230 confers sensitivity to furosemide (Jackel et al., 1998, Thompson et al., 1999a). In the TM2 and TM3 segments residues α1S270 and α1A291 are forming part of binding site for ethanol (Ueno et al., 1999, Krasowski and Harrison, 2000, Ueno et al., 2000), halothane, isoflurane and propofol (Mihic et al., 1997, Krasowski et al., 1997, Krasowski et al., 1998a, Koltchine et al., 1999, Jenkins et al., 2001, Nishikawa et al., 2002, Nishikawa and Harrison, 2003).

Complementing residues of this allosteric site were identified on the α1 subunit TM4 segment. Introduction of a tryptophane mutation in residues α₁Y411, α₁T414 and α₁Y415 was reducing ability of isoflurane, halothane and chloroform to modulate channel function (Jenkins et al., 2002).

A number of residues located on transmembrane domains 1-4 of β subunits are implicated in the formation of recognition sites for compounds discussed above. Residues β₂G219F, β₂N265 and β₂M286, which are homologous to α₁G232, α₁S270 and α₁A291 confer sensitivity to inhaled, general anesthetics and anticonvulsants (Wafford et al., 1994, Thompson et al., 1999, Carlson et al., 2000, Krasowski and Harrison, 2000, Serafiniet al., 2000, Krasowski et al., 2001a,b, Siegwart et al., 2002, Thompson et al., 2002, Chang et al., 2003, Bali and Akabas, 2004).

Findings concerning the β₂N265 residue were recently confirmed in studies of genetically modified mice. Thus, mice carrying β₂N265S knock-in mutation were lacking the sedative effects produced by etomidate (Reynolds et al., 2003) whereas the β³N265S mutation rendered mice insensitive to anesthetic effects of propofol and etomidate, with small reduction in potency of volatile anesthetics (Jurd et al., 2003). However, there are some subtle differences concerning presence of additional sites – for loreclezole and zinc. Selectivity of loreclezole for β₂/β³ over β₁ subunit containing receptors is determined by TM3 residue β₂N289/β³N290. Introduction of a single serine to asparagine mutation in β1 subunit (β1S289N) was enough to confer loreclezole sensitivity of otherwise loreclezole-insensitive GABA_A receptors (Wingrove et al., 1994).

Residues conferring sensitivity of GABA_A receptors to Zn²⁺ were identified in the TM2 domain. Mutations of residues β₂H267 and β₂G270 located close to the entrance of the channel pore were found to reduce inhibition by zinc about 650-fold (Wooltorton et al., 1997a,b, Horenstein and Akabas, 1998, Hosie et al., 2003).

Channel Blockers and their Binding Site

Channel blockers antagonize GABA-elicited currents in a non-competitive fashion (Dillon et al., 1993, Nagata et al., 1994, Nagata and Narahashi, 1994, Ikeda et al., 2001, Huang et al., 2001) and act as convulsants in vivo. Picrotoxinin, U-93631, TBPS and some insecticides are thought to bind at a single binding site located within the channel pore (Xu et al., 1995, Perret et al., 1999, Dibas and Dillon, 2000, Jursky et al., 2000, Buhr et al., 2001).

The binding site of channel blockers is formed by residues located on TM2 segments of both α and β subunits (Figure 1.4.4.2.). Following residues conferring sensitivity to picrotoxinin and TBPS were identified. Residues α₁V257 and α₁T261 were found using the cysteine accessibility method (Xu et al., 1995). Residue α₁V257 was also labelled using site-directed cysteine probes by Perret et al., (1999). Using α₁/β₂ chimeric receptors were β₂A252 and β₂L253 identified residues (Jursky et al., 2000). An additional residue contributing to this binding site β₂T246 located on the linker between TM1 and TM2 segments affects the potency of the convulsant compound pentylenetetrazole (Dibas and Dillon, 2000).

Modulation of GABA_A Receptor Function via Unidentified Allosteric Sites

The majority of compounds acting at the GABA_A receptor discussed in the previous chapters exert their actions via known recognition sites. However, for a number of compounds which are able to allosterically potentiate actions of GABA or directly act on GABA_A receptors structural determinants of their recognition by the receptor have not been identified yet. Neurosteroids is one class of such compounds. They can be divided into two functional groups – uncharged, that can act as positive allosteric modulators (Gee and Lan, 1991, Akk and Steinbach, 2003, Stell et al., 2003) and charged – negative allosteric modulators of receptor function (Zaman et al., 1992, Park-Chung et al., 1999, Akk et al., 2001). Enhancement of submaximal GABA_A receptor currents occurs through increases in both channel open frequency and open duration (Puia et al., 1990, Twyman and Macdonald, 1992, Akk and Steinbach, 2003, Bianchi and Macdonald, 2003). Charged neurosteroids inhibit GABA-gated channel openings by enhancing receptor desensitization and stabilizing desensitized states (Zhu and Vicini, 1997, Shen et al., 2000).

Another group are the γ-butyrolactones and related compounds interacting with the GABA_A receptor, but not at the benzodiazepine or barbiturate sites (Klunk et al., 1982, Mathews et al., 1996). Displacement studies with [^S]TBPS suggested an interaction between the γ-butyrolactones and the picrotoxinin site (Holland et al., 1990a,b,c), however when the picrotoxinin binding site was disrupted by a point mutation potentiation of GABA responses was maintained (Holland et al., 1993, Holland et al., 1995, Williams et al., 1997).

A number of fatty and unsaturated acids were found to modulate GABA_A receptor function. Arachidonic, eicosatetraenoicpentayonic and oleic acids were found to inhibit currents elicited by GABA and muscimol in brain preparations and recombinant GABA_A receptors in dose-dependent manner (Schwartz et al., 1988, Schwartz and Yu, 1992, Saxena, 2000). Thyroid hormones such as L-triiodothyronine (T3) and L-thyroxine are also reported to interact with GABA_A receptors (Chapell et al., 1998), and it has been suggested that the α1-subunit imparts T3 sensitivity (Chapell et al., 1998). The antihelminthic compound ivermectin (Pong and Wang, 1982, Krusek and Zemkova, 1994), the anxiolytic anticonvulsant compounds chlormethiazole and trichloroethanol (Moody and Skolnick, 1989, Hales and Lambert, 1992, Peoples and Weight, 1994), polyamines such as spermine and spermidine (Gilad et al., 1992), and antidepressants such as amoxapine and mianserin (Squires and Saederup, 1988) have been reported to interact with GABA_A receptors but the exact site of action of these drugs and their subunit requirements are not known.

Pharmacology Mediated by GABA_A Receptors in vivo

Different isoforms of the GABA_A receptor differ in their channel kinetics, affinity for GABA, rate of desensitization, subcellular positioning and pharmacology. In the absence of selective pharmacological tools the in vivo function of defined receptor isoform cannot be investigated. Therefore, alternative approaches were used to address this problem. Specific subunit isoforms were either deleted or mutated to alter its properties. Such a deletion or alternation of a subunit isoform would be expected to affect all receptors containing the corresponding subunit isoform. Knockout of individual GABA_A receptor subunits may lead to compensatory upregulation of other subunits. An alternative strategy, which avoids compensatory changes, is the knock-in approach. In this approach, a subunit isoform is mutated such as to alter its pharmacological properties. Transgenic mice were subsequently screened for deficit in the behavioral responses to defined drugs. Thus, allowing conclusions on the in vivo contribution of the GABA_A receptors containing defined subunit isoform.

This strategy helped to understand the in vivo pharmacology of GABA_A receptors containing α₁, α₂, α₃ and α₅ subunits. The point mutation α₁H101R (or equivalent position in other subunits), which renders the mutated subunit isoform insensitive to classical benzodiazepines, has separately been introduced into the different subunit isoforms (Rudolph and Mohler, 2004). A similar approach has been applied to β₂ and β₃ subunit with introduction of β₂N265S and β₃N265S mutations, which render receptors containing mutated subunit isoforms insensitive to general anesthetics (Reynolds et al., 2003, Jurd et al., 2003).

Pharmacological Properties Mediated by the α Subunits

GABA_A receptors containing the α₁ subunit are the most abundant and expressed in all brain areas. Several studies were undertaken to elucidate functions mediated by receptors containing α₁ subunit, using both the knock-out and knock-in approaches. Mice with a deleted gene coding for the α₁ subunit developed normally and it was found that ablation of this particular subunit was compensated by overexpression of α₂ and α₃ subunits enough to sustain function of GABAergic inhibitory system (Sur et al., 2001, Kralic et al., 2002a,b, Goldstein et al., 2002). This deletion caused developmental changes (Vicini et al., 2001) and reduced sensitivity of mutant mice to the locomotor-stimulating effects of ethanol (Kralic et al., 2003).

In mice carrying α₁ subunit containing receptors in which α₁H101R mutation had been introduced diazepam lost its ability to mediate sedation (Rudolph et al., 1999, Crestani et al., 2000a,b, Low et al., 2000, McKernan et al., 2000). Additionally α₁-containing receptors were found to mediate the amnestic and anticonvulsant activity of diazepam (Rudolph et al., 1999, Crestani et al., 2000a). Mice carrying the α₂H101R point mutation lost the anxiolytic effect of diazepam (Low et al., 2000). This lack of response was specific for ligands of the benzodiazepine site, since α₂H101R mice retained the ability to display an anxiolytic-like response to sodium pentobarbital. Thus, the anxiolytic action of diazepam is selectively mediated by the enhancement of GABAergic transmission in a population of neurons expressing the α₂ subunit containing GABA_A receptors (Low et al., 2000). Additionally α₂ subunit containing GABA_A receptors were found to mediate the muscle relaxant effect (Crestani et al., 2001). The analysis of mice carrying the α₃H126R mutation indicated that the anxiolytic effect of benzodiazepine drugs is not mediated by α₃-receptors (Low et al., 2000). However, α₃ subunit containing receptors seem to be implicated in muscle relaxant effect of diazepam, but only at high doses (Crestani et al., 2001).

The native α₄ subunit containing receptors in the brain are associated with actions of the neurosteroids (Mihalek et al., 1999, Spigelman, 2002, Spigelman, 2003, Stell et al., 2003), implicated in actions of alcohol (Mihalek et al., 2001, Sundstrom-Poromaa et al., 2002, Wallner et al., 2003) and formation of alcohol-dependence (Mahmoudi et al., 1997, Follesa et al., 2003). In steroid-withdrawal models of premenstrual syndrome and postpartum or postmenopausal dysphoria, particularly the increased anxiety and incidence of seizures was also attributed to α₄ subunit containing receptors (Smith et al., 1998, Follesa et al., 2000, Gulinello et al., 2001, Hsu and Smith, 2003, Gulinello et al., 2003a,b).

Two transgenic models have been generated to study contribution of α₅ subunit. In one the entire subunit has been deleted (Collinson et al., 2002), and in the second the α₅H105R point mutation has been introduced (Crestani et al., 2002). Both of these genetically modified mice showed an improved performance in animal models of learning and memory (Collinson et al., 2002, Crestani et al., 2002), suggesting that a selective inhibitor of α5 subunit containing receptors could have use as a cognitive enhancer, for instance in mild cognitive impaired elderly, or Alzheimer’s disease patients.

Studies on knockout mice that lack the α6 subunit reported no change in the response of these mice to pentobarbital, general anesthetics or ethanol, compared with wild-type mice (Homanics et al., 1997a), but the knockout mice were more sensitive to the motor-impairing action of diazepam (although in a limited dose range only) than their wild-type counterparts (Korpi et al., 1999). In addition, a selective post-translational loss of the δ subunit was apparent in cerebellar granule cells, which indicates that the δ subunit is co-assembled with the α6 subunit (Jones et al., 1997). The absence of the α6 subunit triggered various additional changes in the cerebellum, which included a reduction in the affinity of the GABA_A receptor for muscimol (Homanics et al., 1997a), an increase in the number of receptors containing the β3 subunit compared with wild-type (Nusser et al., 1999b) and, interestingly, a compensatory upregulation of a K+ channel (TASK-1) in granule cells (Brickley et al., 2001).

Pharmacological Properties Mediated by the β Subunits

GABA_A receptors that contain the β₂ and β₃ subunits are a prevalent receptor population present in most brain areas (Fritschy and Mohler, 1995). It was observed that expression of β subunits is altered in patients with temporal lobe epilepsy (Brooks-Kayal et al., 1998, Loup et al., 2001, Pirker et al., 2003) and also in various experimental models of epilepsy (Tsunashima et al., 1997, Schwarzer et al., 1997). Deletion of the gene encoding the β₃-subunit results in mice that possess only half of the normal density of GABA_A receptors in the brain (Krasowski et al., 1998b). Most of these mice die in the neonatal period; however, a few survive and grow to normal body size (Homanics et al., 1997b), although these mice display various neurological impairments including hyperresponsiveness to sensory stimuli (Ugarte et al., 2000), strong motor impairment and epileptic seizures (DeLorey et al., 1998), which might be due to the lack of β₃-containing receptors as ‘desynchronizers’ of neuronal activity (Huntsman et al., 1999, Ramadan et al., 2003).

The sedative and anesthetic effects of anesthetics were also found to be mediated via GABA_A receptors composed from different β subunit isoforms (Quinlan et al., 1998, Laposky et al., 2001, Wong et al., 2001). Mice carrying β₂N265S mutation were lacking the sedative effects produced by etomidate (Reynolds et al., 2003). In another study β₃N265S mutation rendered mice insensitive to anesthetics propofol and etomidate, suggesting that it has a key role in mediating the hypnotic and immobilizing responses in vivo. Volatile anesthetics showed only small reduction in their effects and appear to act via a broader spectrum of molecular targets (Jurd et al., 2003).

Pharmacological Properties Mediated by the γ Subunits

Mice deficient in both the γ_2S and γ_2L subunits are entirely devoid of a response to benzodiazepines as shown behaviorally and in cultured dorsal root ganglion cells (Gunther et al., 1995). Most homozygous γ₂ knockout mice die perinatally. This is due, at least in part, to the requirement of the γ₂ subunit for synaptic clustering of GABA_A receptors, although not for receptor assembly (Essrich et al., 1998). In animals that survive for up to two weeks, diazepam failed to induce sedation and to impair the righting reflex. This failure reflects the requirement of the γ₂ subunit for the formation of the benzodiazepine site of GABA_A receptors (Gunther et al., 1995, Sigel, 2002). By contrast, mice heterozygous for the γ₂ subunit knockout mutation develop and behave normally. The synaptic clustering of GABA_A receptors is only partly reduced (~15–30%, depending on the brain region); the unclustered receptors consist of α and β subunits. When exposed to certain fear-inducing stimuli, these animals show a striking disease phenotype with a high anxiety response to natural and learned aversive stimuli, as well as a cognitive bias for threat cues (Crestani et al., 1999).

Pharmacological Properties Mediated by the δ Subunit

Disruption of the gene encoding the δ subunit produced mice with an epileptic phenotype (Spigelman et al., 2002), changes in expression of α4 and γ2 subunits in the forebrain (Peng et al., 2002, Korpi et al., 2002) and cerebellar granule cells (Tretter et al., 2001). δ subunit knockout mice displayed an attenuation of the sleep time following the administration of the neurosteroids alphaxalone and pregnenolone and ethanol, whereas the response to propofol, etomidate, ketamine and midazolam was indistinguishable from that observed in wild-type mice (Quinlan et al., 2000). Behavioral responses of δ deficient mice to neurosteroids and ethanol were also greatly altered, suggesting important role of this subunit type in endogenous functional modulation of δ subunit containing GABA_A receptors (Mihalek et al., 2001). This behavioral changes may be attributed to reduced sensitivity to neurosteroids in hippocampus (Spigelman et al., 2003), thalamic relay neurons (Porcello et al., 2003) and cerebellum (Vicini et al., 2002).

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External links

• Basic Neurochemistry: GABA Receptor Physiology and Pharmacology