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Home , DMCM, Flumazenil, GABA receptor, GABA receptor antagonist, Isoguvacine, Phaclofen

GABA RECEPTORS

Ian L Martin and Susan M J Dunn receptors. The became known as GABAC . The DNAs that encode these receptor Ian Martin is Professor of in proteins have now been identified, providing not the School of Life and Health Sciences at the only a facile means for their molecular University of Aston, Birmingham B4 7ET, UK. characterisation but also a significant stimulus for our attempts to understand their Susan Dunn is a Senior Lecturer in the physiological importance. Department of Pharmacology at the University of Bristol, University Walk, Bristol The GABA Receptors BS8 1TD, UK. They have common interests A in GABAergic transmission in the central The GABAA receptors are widely distributed nervous system and their research is focused within the mammalian CNS and exhibit a on the structure-function relationships of the differential topographical distribution.9 Group 1a ligand-gated receptors. Systematic modification of the natural agonist demonstrated that GABAA receptors can be activated by a number of compounds (Figure 1) Historical Perspective such as , , 3- aminopropane sulphonic acid, piperidine-4- GABA is the major inhibitory amino acid sulphonic acid and 4,5,6,7-tetrahydro-[5,4-c]- 10 transmitter of the mammalian central nervous pyridin-3-ol, many of which were subsequently 11 system and it is present in some 40% of all used as . At equilibrium the neurones. Most of the early studies, carried out binding of GABA agonists is heterogeneous with with iontophoretic application of GABA in the a high affinity component (Kd values of 10-20 CNS, indicated that it generally produced nM) and one or more low-affinity sites with inhibitory hyperpolarizing responses on dissociation constants in the range of 100 nM to 12 neurones,1 which were blocked competitively by 1m M. The presence of even lower affinity sites the alkaloid .2 The hyperpolarizing (Kd values about 50m M) has been inferred from response is due to an increase in the chloride conductance of the neuronal membrane Figure 1. Structures of selected GABA receptor allowing chloride ions to flow down their agonists electrochemical gradient into the cell.3,4 However, in the late 1970s, Bowery and his H NCOH colleagues, in attempts to identify GABA 2 2 GABA (Cat. No. 0344) CO H receptors on peripheral nerve terminals, noted OH 2 that GABA application reduced the evoked H N release of noradrenaline in the rat heart and that 2 N this effect was not blocked by bicuculline. This O N H action of GABA was mimicked by , 4- Muscimol (Cat. No. 0289) Isoguvacine (Cat. No. 0235) amino-3-(4-chlorophenyl) butanoic acid (Figure 1), a compound that had no effect on chloride O H NCOH conductance in central neurones. The new 2 2 - + O receptor was named GABA to differentiate it CACA (Cat. No. 0179) H3N B HH from its more familiar cousin, which was termed Cl (1SR ,2 )-(+)-2-(aminomethyl)- 5,6 GABAA . The GABAC receptor had a rather cyclopropane-1-carboxylate more difficult birth. In an attempt to discover which conformation of GABA was responsible H O + - for activating the receptor, Johnson and his H3N P O H2N colleagues7 synthesised a number of OH O H 3-aminopropyl phosphinic acid conformationally restricted analogues of GABA (R )-Baclofen (Cat. No. 0796) and noted thatcis -4-aminocrotonic acid (CACA), HO C NH which has a partially folded conformation (Figure 2 2 O 1), depressed the firing of cat spinal neurones in H2NP a bicuculline insensitive manner. These Me OH depressant effects could not be reproduced by SKF 97541 (Cat. No. 0379) 8 baclofen, suggesting a pharmacology distinct (Cat. No. 0806) from that of either the GABAAB or GABA (Bold text denotes compounds available from Tocris)

Tocris Cookson Ltd., UK Tocris Cookson Inc., USA Tel: + 44 (0)117 982 6551 Tel: (800) 421-3701 Fax: + 44 (0)117 982 6552 www.tocris.com Fax: (800) 483-1993 e-mail: [email protected] [email protected] e-mail: [email protected] the higher concentrations of GABA that are The purification of the receptor protein in the required to activate the channel. There has been early 1980s29 provided the opportunity to raise much speculation about the role of these sites in monoclonal antibodies to the receptor, which receptor function. Whether the sites are were later used to study the fine anatomical physically distinct or represent allosteric detail of receptor distribution.28 Subunit conformations of the same site remains an purification, and elucidation of their partial amino intriguing question.13 acid sequences was crucial in their eventual molecular cloning. Only two receptor subunits Electrophysiological studies demonstrated that (ab and ) were initially identified at the protein the activation of the receptor resulted in level and, in the mid 1980s these were cloned. increased chloride conductance of the cell When co-expressed from their encoding nucleic membrane3,4 with the concentration-response acids inXenopus oocytes, these subunits curve exhibiting positive cooperativity, consistent produced a receptor that responded to GABAA with the presence of at least two agonist binding receptor agonists and antagonists in the sites on the receptor molecule.14-16 The agonist expected manner.30 induced current decreased on continued exposure to high agonist concentrations17-19 Subsequent studies have revealed a multiplicity suggesting that these receptors undergo of protein subunits that have been divided into desensitisation. Biophysical characterisation of seven classes, according to similarities in their the receptor, initially using noise analysis of deduced amino acid sequence. Within these neurones in primary culture, provided the first classes there are further subdivisions into estimates of mean single channel conductance subunit isoforms, some of which exhibit and average channel open times.20 The latter alternate splice variants. In man, sixab , three varied with the nature of the activating agonist.21 and threeg subunit isoforms are presently Development of single channel recording known, together with single representatives of techniques22 provided further detail on the thedep , , and q classes. Additional isoforms in stochastic nature of the single channel events other species are known for some of these with the demonstration of multiple single classes.31 Within a single subunit class the channel conductances: 44, 30, 19 and 12 pS.23 sequence homology is about 70% but between The 30 pS conductance is the most prevalent classes this falls to around 30%. Deduced with distinct open time states, varying from 0.5 amino acid sequences of subunits from all of the to 7.6 ms. The distribution of these states is families indicate that each has a long amino agonist concentration dependent, with longer terminus of about 200 amino acids which open times predominating at higher agonist contains a signature -cysteine loop, concentration.24,25 The competitive antagonist common to the receptor family, prior to the first bicuculline appears to reduce conductance of four hydrophobic segments of about 20 amino through the channel by reducing not only the acids, which are termed TM1-TM4. TM2 is opening frequency but also the mean open thought to form a major part of the lining of the time.24 Other competitive antagonists, such as ion channel as it crosses the cell membrane, the pyridazinyl GABA derivative SR 95531 while between TM3 and TM4 there is a large (Figure 2), are available. In addition, the intracellular loop, which is the most divergent receptor can be blocked non-competitively by part of the sequence within the sub-family. The and by a number of GABAA receptors have been purified from the bicyclophosphates.26 also decreases pig brain and imaged, after negative staining, in the channel open probability in a manner that is the electron microscope.32 The receptor has a compatible with open channel block.27 diameter of about 8 nm in the plane of the membrane and consists of five protein subunits Figure 2. Structures of selected GABA receptor arranged pseudosymmetrically around the antagonists integral ion channel; it appears very similar to NH images of the nicotinic . O OH NMe N O Despite the plethora of receptor subunits, it HH N O

O appears that there are a limited number of combinations expressedin vivo .33 A separate O gene encodes each subunit and in situ O O OMe hybridisation, together with immuno- (+)-Bicuculline (Cat. No. 0130) SR 95531 (Cat. No. 1262) histochemical studies, have revealed a distinct distribution for these gene products,33,34 Me consistent with the idea that they each serve a HN P OOH defined physiological role. The recognition and TPMPA (Cat. No. 1040) functional characteristics of the individual OH GABAA receptor subtypes are defined by their CO2H PO(OH)2 constituent subunits and while ectopic Me Cl N Me H expression studies continue to explore this NH2 SCH 50911 (Cat. No. 0984) diversity, the authentication of particular GABAA (Cat. No. 0178) receptor subtypesin vivo remains a significant HN P task. However, it is clear that the most common HO O OH O OH Me H2NPOEt GABAA receptor in the mammalian CNS

Cl consists of two copies each of theab 1 and 2 OEt subunits together with a singleg 2 subunit.35 It is CGP 35348 (Cat. No. 1245) Cl CGP 55845 (Cat. No. 1248) this receptor that appears to mirror the pharmacological and biophysical characteristics (Bold text denotes compounds available from Tocris)

2 of the GABAA receptors characterised previously Figure 3. Viewing the GABAA receptor from the in whole animals and in primary cultures of extracellular space, the orientation of the central neurones. subunits within the pentamer together with the location of the (Bz) and low Weiss and colleagues36 used site-directed affinity GABA sites shown is in accord with all the mutagenesis data that is available to date mutagenesis to identify residues in the GABAA receptor that are involved in channel activation. They identified residues in theb subunit that Bz appear to be important for low affinity agonist recognition. Based on homology with other members of the receptor family, and the currently available evidence from labelling and g2 a1 mutagenesis studies, it appears likely that the GABA activation sites for GABA are located at theba - interface(s). Recent work has identified residues in theb subunit that may be involved in high affinity agonist binding and this has led to the b2 b2 suggestion that these site(s) may lie at theab - andgb - interfaces.13 The current consensus is that a benzodiazepine modulatory site lies at the a1 interface betweenag and subunits.37 Much of GABA the evidence for binding site locations is somewhat indirect, involving mutational studies to selectively disrupt the sites. However, our ability to interpret these data has recently been , , muscle relaxant and significantly enhanced by the crystallisation of amnesic, are produced via the GABAA an acetylcholine binding protein which bears a receptors. However, not all the GABAA receptors marked structural similarity to the extracellular that exist in the brain recognise the domain of this receptor family.38 This protein, . The particulara subunit though not a ligand-gated ion channel, displays isoform present within an individual GABAA remarkable homology within its ligand binding receptor subtype is the primary determinant of domains. The crystal structure thus provides a benzodiazepine recognition (Table 1). If thea 1 valuable physical template against which the subunit of the most common GABAA receptor is mutational information may be interpreted. replaced withaa 4 or 6, the receptor fails to Figure 3 shows the subunit arrangement of the recognise the benzodiazepines. It is now clear from both biochemical and mutational analysis GABAA receptor that is consistent with the currently available ligand recognition site data. that this insensitivity is due primarily to a single amino acid substitution: an arginine residue in The Benzodiazepines aa4 and 6 subunits replaces histidine 101, which is present inaaa 1, 2, 3 and a 5 subunits.42,43 While receptors containinga 1, The benzodiazepines, because of their aa2, 3 or a 5 together with a b and a g subunit therapeutic importance, have been a major are all recognised by the ‘classical’ focus of GABA receptor research since the A benzodiazepines, several agents are able to discovery of saturable, high affinity binding sites distinguish the receptors on the basis of their a for [3 H] in the CNS. 39,40 Agonist subunit isoform composition. The first of these activation of the GABA receptor is augmented A compounds was the triazolopyridazine CL218, by the anxiolytic benzodiazepines, causing a 872 ,44 which is related to the recently parallel leftward shift of the GABA introduced hypnotic ‘’ (Figure 4), while concentration-response curve. All the overt certainb -carboline-3-carboxylic acid esters effects of the benzodiazepines: ,

Table 1. Approximate binding affinities of benzodiazepine site ligands for GABAA receptor subtypes. Compoundabg 1 2 abg22 abg 32 abg 42 abg 52 abg 62 Diazepam16.1 16.9 17.0 > 10,000 14.9 > 10,000 1.3 1.7 2.0 – – > 10,000 1.8 1.2 3.0 – 1.2 – 1.2 1.2 1.3 – 2.4 – Ro15-1788 () 1.0 1.1 1.5 107 0.4 90 Ro15-4513 2.6 2.6 1.3 – 0.24 6.5 CL218872 130 1820 1530 > 10,000 490 > 10.000 b-CCM 1.7 6.5 4.1 – 27 2050 17 291 357 – > 15,000 – L-655,708 48.5 27.4 24.5 – 0.45 83.2

(Bold text denotes compounds available from Tocris) Values are quoted in nM. Information abstracted from references 41, 89 and 90. Note that the determinations were carried out with eitherbb 2 or 3 subunit isoforms, which do not have a pronounced effect on the affinity of benzodiazepine site ligands.

3 Figure 4. Structures of selected benzodiazepine agonists.49,50 Indeed, electrophysiological site ligands experiments with the inverse agonists in vitro

CN show that they shift the GABA concentration- CH3 O H3C N N N response curve to the right, thus decreasing the N potency of the natural transmitter. While the N N Cl N agonist benzodiazepine site ligands increase Cl N O channel opening frequency, the inverse agonists F decrease it.51 It is now clear that within this N CH3 series ofb -carbolines, whereas the ethyl ester is Diazepam CH3 pro- and thus a partial inverse Zaleplon agonist, the propyl ester is essentially devoid of O 52 N N efficacy and thus an antagonist. At the other N Cl end of the scale a methyl ester analogue, 6,7- NMe N O N dimethoxy methylb -carboline-3-carboxylate O Me NMe2 (DMCM), is overtly convulsant and thus a full N O inverse agonist. Zolpidem (Cat. No. 0655) N Me (Cat. No. 1094) O Attempts to delineate the functional importance N of individual GABA receptor subunits using the OEt A N H gene knockout technology have proved less than fruitful with thebg 3 and 2 proving N neonatally lethal53,54 while thea 6 knockout MeO O mouse displayed no overt phenotype.55 O L-655,708 (Cat. No. 1327) N However, a knock-in approach, which makes OEt use of the histidine/arginine exchange of the a CO Pr N 2 subunits mentioned above, has demonstrated N NMe N that particular GABAA receptor subtypes H F mediate distinct aspects of benzodiazepine O b-CCP (Cat. No. 0612) Flumazenil (Cat. No. 1328) pharmacological profile: those receptors containing thea 1 subunit are responsible for (Bold text denotes compounds available from Tocris) the sedative effects while those containing the a2 subunit are responsible for their anxiolytic allowed a similar distinction (Figure 4).45 effects.56 This knowledge will undoubtedly prove Zolpidem (Figure 4), currently the most widely valuable in the development of agents with a prescribed hypnotic in the USA, is also able to restricted pharmacological profile and a recent distinguish GABAA receptors on the basis of review of the patent literature suggests that this theira subunit isoform composition: it has a approach is well advanced.57 high affinity for those receptors which contain a1, a lower affinity for those receptors which Other Allosteric Sites containaa 2 or 3 and a very low affinity for those receptors which containa 5.46 Again The also produce many of their zolpidem does not recognise receptors which effects by interaction with the GABAA receptors containaa 4 or 6 subunits.47 A nomenclature of the mammalian CNS. Like the for these receptors with distinct affinities for the benzodiazepines, they shift the GABA ‘subtype selective’ ligands was developed prior concentration-response curve to the left but to the cloning era:a 1 containing receptors unlike the agonist benzodiazepines, the proved to have the Bz1 phenotype, while those barbiturates also increase the maximum that containedaa 2, 3 or a 5 subunits exhibited response. They clearly interact with a distinct Bz2 pharmacology. Other nomenclatures have allosteric site; the barbiturates augment the appeared during the intervening years GABA mediated current by increasing the culminating, most recently, in the classification average channel open time but have little effect 58,59 of GABAA receptors by a combination of on channel opening frequency. Whereas the molecular and pharmacological benzodiazepines require the presence of a g 48 characteristics; time will tell if the scientific subunit within the GABAA receptor oligomer to community finds it acceptable. exert their effects60 this is not the case for the barbiturates. It is also clear that the barbiturates, Perhaps one of the most interesting at high concentrations, are able to open GABAA phenomenological observations to appear from receptor channels directly, which also studies of the benzodiazepine interaction with distinguishes them from the 61,62 the GABAA receptors has been the development benzodiazepines. of the inverse agonist concept. The classical benzodiazepines are anxiolytic, sedative/ In addition to allosteric sites for the hypnotic, anti-convulsant and muscle relaxant benzodiazepines and barbiturates, the GABAA but one of the first non-benzodiazepine ligands receptors also exhibit high affinity recognition discovered, which was able to displace the sites for certain steroids. The observation that benzodiazepines from their binding sites, was alphaxalone, the synthetic steroid general ethylbb -carboline-3-carboxylate ( -CCE). This anaesthetic, was able to cause stereoselective compound has effects which are diametrically potentiation of GABAA receptor mediated opposed to those of the classical responses in cuneate nucleus slices from rat benzodiazepines, i.e. it is pro-convulsant. It was brain63 was subsequently confirmed in voltage termed an inverse agonist with the classical clamp studies conducted in both neuronal and benzodiazepines being then classified as adrenomedullary chromaffin cells.64,65 The

4 Figure 5. Structures of selected steroids, together with exhibiting the expected barbiturates and anaesthetics that interact with stereospecific effects.70 The volatile the GABAA receptor anaesthetics, the halogenated ethers and alkanes together with and diethyl H C 3 O ether are differentiated from the intravenous anaesthetics such as the barbiturates, and by their mechanism of action, but H3C O both groups appear to facilitate GABA mediated HO H inhibition at the GABAA receptor. While there remains a good deal of work to do, studies over 3aa -hydroxy-5 -pregnan-20-one HO the past decade have provided a significant H body of evidence to address the sites on the 3ab -hydroxy-5 -pregnan-20-one receptor protein which form the targets for HO O interaction with the anaesthetics. Not surprisingly these agents appear to bind to hydrophobic pockets within the protein although differences have been identified with, for HO H HO example, a specific aspartate residue within Allotetrahydrodeoxycorticosterone 3aa ,21-dihydroxy-5 -pregnan-20-one TM2 being required for etomidate sensitivity but of no consequence to the activity of

HO pentobarbitone, propofol or the anaesthetic H 71 F steroids. F Tetrahydrodeoxycorticosterone O F 3ab ,21-dihydroxy-5 -pregnan-20-one H H Cl F F Although the majority of studies have focussed O Enflurane on the GABAA receptor it is clear that certain O anaesthetics, such as ketamine, nitrous oxide OH O O and xenon do not produce their effects through NN this receptor but probably by inhibition of theN - HO H OH methyl-D -aspartate receptor. It is also clear that Propofol (Cat. No. 0939) Alphaxolone Pentobarbitone many of the anaesthetics interact with other ligand gated ion channels, in addition to the (Bold text denotes compounds available from Tocris) NMDA receptor, with pronounced effects being seen on the neuronal nicotinic acetylcholine progesterone metabolites 3aa -hydroxy-5 - receptors, particularly those containing thea 4 pregnan-20-one (allopregnanolone) and 3a - subunit, and the 5-HT3 receptor. Undoubtedly hydroxy-5b -pregnan-20-one (pregnanolone) the potential target population will expand as 72 together with 3aa ,21-dihydroxy-5 -pregnan-20- these studies progress. one (allotetrahydrodeoxycorticosterone) are even more potent than alphaxalone (Figure 5). It is GABAC receptors now clear that these steroids facilitate GABA mediated responses via recognition sites distinct In addition to the GABAA receptors there is a from both the barbiturates and the distinct class of ligand gated ion channels that benzodiazepines. Mechanistically the active are activated by GABA; referred to as the 8 steroid analogues appear to produce their effects GABAC receptor. The natural agonist GABA is by increases in both channel open time and about an order of magnitude more potent at the opening frequency; like the barbiturates the GABAC receptors than at the most common of the GABA receptors. The GABA receptors are steroids directly activate GABAA receptor AC mediated channels at high concentrations.66 The activated bycis -aminocrotonic acid (CACA), which is not recognised by either the GABA or neuroactive steroids show limited GABAA A receptor subtype specificity. However, it appears GABAB receptors, suggesting that they that in those receptors containing thea 4 subunit, recognise the partially folded conformation of GABA currents are inhibited by the naturally GABA (Figure 1). GABAC receptors are not occurring pregnanolone and alphaxalone while blocked by bicuculline and do not recognise the benzodiazepines, barbiturates or the they are stimulated in other GABAA receptors. Thed subunit also appears to markedly increase neuroactive steroids but, like GABAA receptors the efficacy of both pregnanolone and are blocked by picrotoxin, while 1,2,5,6- alphaxalone inabd 4 3 receptors compared to tetrahydropyridine-4-yl methyl phosphinic acid those comprised ofabg 4 3 2 subunits.67 These (TPMPA; Figure 2) appears to inhibit GABAC observations are particularly interesting in view receptors selectively. Pharmacologically they of the plasticity of bothad 4 and subunit are thus quite distinct. However, molecular expression associated with pregnancy,68,69 cloning studies have revealed that this observations which may prove to have a bearing pharmacological profile is remarkably similar to on the mood changes associated not only with that exhibited by ther subunits when expressed premenstrual stress but also those experienced ectopically.73 Two homologousrr subunits, 1 in postpartum depression. andr 2, have been identified in man and these can be expressed as homomers or heteromers, but do not co-assemble with any of the GABA There is now general consensus that the GABAA A receptor plays a significant role in general receptor subunits. The DNAs are encoded on anaesthesia. These receptors fulfil the necessary chromosome 6 in man, distinct from the clusters criteria being sensitive to clinically relevant of GABAA receptor subunit genes which are concentrations of the anaesthetic agents found on chromosomes 4, 5, 15 and X; the r subunits are between 30 and 38% homologous

5 to the GABAA receptor subunits at the amino active isomer (Figure 1). There is evidence that acid level. In the important TM2 region of the GABAB receptor agonists may be useful in the sequence, they show greater homology to the treatment of pain and to reduce the craving for glycinea subunits than to any of the GABAA of addiction. There is limited information receptor subunits. It is assumed that they form on the therapeutic potential of GABAB receptor pentameric assemblies, similar to the other antagonists but there is support for the idea that members of the ligand gated ion channel family, they may prove valuable in the treatment of which enclose a chloride selective channel. The absence and as cognition enhancers.76 single channel conductance of the homomeric or heteromeric receptors composed ofr subunits GABAB receptors have now been identified by is smaller (around 7 pS) than that exhibited by expression cloning using the high affinity ligand 77 the GABAA receptors (25-30 pS) and the gating CGP-64213. Functional receptors are formed kinetics are quite distinct, with both the only after heterodimerization of GABA(B1) and activation and deactivation time constants being GABA(B2) (previously known as GBR1 and very slow. Theser homomers or heteromers GBR2) by interaction through their C-termini, the also appear to be remarkably resistant to first time that this form of 1:1 stoichiometry has desensitisation in the presence of high been identified within this family.78-81 Both concentrations of the agonist. While it is clear subunits are members of the 7-transmembrane that the homomeric and heteromeric receptor family that show over 30% sequence combinations of ther subunits display distinct homology to the metabotropic glutamate biophysical and recognition characteristics74 receptors. A number of splice variants have they do generally mirror, quite closely, the been identified for both GABA(B1) and 82 GABAC receptors that have been characterised GABA(B2) . The mRNA encoding GABA(B2) is in retinal bipolar cells from a number of species. found exclusively in neurones but GABA(B1) is Discussions continue with regard to the found in both neurones and glia. nomenclature: do we use sequence homology to Immunoprecipitation studies suggest that classify proteins or their functional and GABA(B1) and GABA (B2) are always found as recognition characteristics.75 This question will heterodimers although the paucity of mRNA for not easily be resolved: a man with one watch GABA(B2) in the striatum has led to suggestions knows what time it is but a man with two that further subunits remain to be identified.82 watches is never quite sure! There have been suggestions that the two most widely studied splice variants GABA(B1a) and GABAB Receptors GABA(B1b) my be differentially located within the cell, the former being pre-synaptic while the latter is found post-synaptically. The large GABA also activates metabotropic GABAB receptors, which are widely distributed within the deletion at the N-terminus which occurs in splice and also in peripheral variant GABA(B1b) is consistent with a differential autonomic terminals.5 Their activation causes an subcellular localisation and it will be interesting inhibition of both basal and forskolin stimulated to see the whether or not this is region specific. adenylate cyclase activity together with a decrease in Ca2+ and an increase in K + Although a significant number of phosphinic acid conductance in neuronal membranes. The derivatives have been synthesised as GABAB receptors are activated by baclofen, used in the receptor agonists few exceed the potency of treatment of spasticity, (R )-baclofen being the (R )-baclofen and none has proved useful in the

Table 2. Comparative pharmacology of GABA receptors

Compound GABAABC GABA GABA Reference GABA Agonist Agonist Agonist Muscimol Agonist Inactive Partial agonist 6, 8, 91 Isoguvacine Agonist Inactive Antagonist 6, 8 THIP Agonist Inactive Antagonist 6, 8 P4S Agonist Inactive Antagonist 6, 8 TACA Agonist Inactive Agonist 8 CACA Inactive Inactive Partial agonist 8 (R )-Baclofen Inactive Agonist Inactive 6, 8 Bicuculline Antagonist Inactive Inactive 6, 8 Picrotoxin Antagonist Inactive Antagonist 6, 8 CGP 35348 Inactive Antagonist Inactive 76 CGP 54626 Inactive Antagonist Inactive 76 CGP 64213 Inactive Antagonist Inactive 76 SCH 50911 Inactive Antagonist Inactive 76 TPMPA Inactive Inactive Antagonist 8, 86, 87

(Bold text denotes compounds available from Tocris)

6 differentiation of the distinct receptor subtypes. However, this has been compromised, until However, recently it has been reported that recently, by the paucity of potent, systemically gabapentin selectively activates the GABA(B1a,2) active and selective ligands. The molecular heterodimer coupled to inwardly rectifying cloning of their constituent cDNAs coupled with potassium channels expressed in Xenopus the discovery that they function as heterodimers oocytes but this did not occur with GABA(B1b,2) or has now engendered a new urgency to research 83 GABA(B1c,2) . While the early specific GABAB in this arena and the future promises to be receptor antagonists suffered from a limited exciting. potency with phaclofen, for example, displaying an affinity of only 100m M, a number of selective, Conclusions high affinity and systemically active antagonists are now available, although most rely on the Interest in the receptors for GABA, the major phosphinic acid moiety (Figure 2). It is inhibitory transmitter in the CNS, has been undoubtedly only a matter of time before developed, with varying degrees of enthusiasm, subtype specific antagonists will become over the past 40 years. We now have agonists available. and antagonists which allow us to differentiate, experimentally at least, between responses Like the metabotropic glutamate receptors, both mediated by the three pharmacologically distinct the GABA(B1) and GABA (B2) subunits possess a receptor families with which they interact (Table large extracellular N-terminal tail which has 2). The information base is most extensive for been modelled by homology on the bacterial the GABAA receptors, driven largely by periplasmic binding protein motif characterised observations that these proteins are the targets by two globular domains connected by a hinge for a number of drugs with significant clinical region. It has been suggested that agonist importance. The expansion continues with the activation relies on the closure of the two conviction that this almost bewildering globular domains subsequent to agonist binding: complexity can be harnessed for the next the venus fly-trap model.84 A limited number of generation of pharmacological agents with a mutagenic studies have been carried out to more restricted profile of activity. The GABAB delineate the residues important in agonist and receptors remain the rather poor cousins in the antagonist recognition85 but the nature of the sense that their potential is not yet realised. The interaction between the two proteins within the developments over the past 7 years or more heterodimer, in both ligand recognition and their have delivered a promissory note that is liaison with the G-proteins integral to receptor producing significant investment and many hold function remains to be investigated. real conviction in their future. Surveys of the literature would suggest that the GABAC The GABAB receptors remain somewhat forlorn. receptors have generated rather more interest Their ubiquitous distribution within the CNS, from the nomenclaturists than is warranted. their proven importance in the modulation of However, recent evidence that their distribution transmitter release and the late inhibitory is more diverse than previously thought88 could postsynaptic potential promise a good deal as provide a further extension to the potential targets for pharmacological intervention. inhibitory control of the central nervous system.

References 45.Nielsen and Braestrup (1980) Nature286 606. 46 . Pritchett and Seeburg(1990) J.Neurochem.54 1802.47 . Hadingham et al (1993) Mol.Pharmacol.43 970.48 .Barnard et al (1998) 12.Krnjevic and Schwartz (1967) Exp.Brain Res.3 320. . Curtis et Pharmacol.Rev.50 291.49 .Tenen and Hirsch (1980) Nature 288 al(1970) Nature226 1222.3 .Curtis et al (1968) Exp.Brain Res. 5 1. 609.50 .Cowanet al (1981) Nature290 54. 51 . Barker et al (1984) 45.Kellyet al (1969) Exp.Brain Res.7 11. . Bowery et al (1981) in Actions and interactions of GABA and the benzodiazepines, p203, Eur.J.Pharmacol.71 53.6 .Hill and Bowery (1981) Nature 290 149. Publ. Raven.52 .Smiechen et al (1993) in Anxiolyticb -carbolines: 78.Johnstonet al (1975) J.Neurochem.24 157. . Johnston (1996) from molecular biology to the clinic, p7, Springer Verlag.53 . Culiat et TiPS17 319.9 .Palacios et al (1981) Brain Res 222 285. al(1995) Nature Genet.11 344.54 .Gunther et al (1995) Proc.Natl. 10.Krogsgaard-Larsen et al (1984) in Actions and interactions of Acad.Sci.USA92 7749.55 .Nusser et al (1999) Eur.J.Neurosci. 11 GABA and the benzodiazepines, p109, Publ Raven.11 . Stephenson 1685.56 .Ruddolphet al (2001) TiPS22 188. 57 . Martin and (1988) Biochem.J.249 21.12 .Fisher and Olsen (1986) in Lattman(1999) Exp.Opin.Ther.Patents9 1347.58 . Study and Benzodiazepine/GABA chloride channels: structural and functional Barker(1981) Proc.Natl.Acad.Sci.USA78 7180.59 . Twyman et al properties, p241, Alan R. Liss.13 .Newell et al (2000) J.Biol.Chem. (1989) Ann.Neurol.25 213.60 .Pritchett et al (1989) Nature 338 27514198.14 .Dichter (1980) Brain Res. 190 111. 15 . Sakmann et 582.61 .Nicollet al (1975) Nature258 625. 62 . Simmonds (1981) al (1983) J.Neural.Transm.suppl.18 83.16 . Bormann and Clapham Br.J.Pharmacol.73 739.63 .Harrison and Simmonds (1984) Brain (1985) Proc.Natl.Acad.Sci.USA82 2168.17 . Adams and Brown Res.323 287.64 .Cottrell et al (1987) Br.J.Pharmacol. 90 491. (1975) J.Physiol.250 85.18 .Akaike et al (1987) J.Physiol. 391 219. 65.Harrisonet al (1987) J.Neurosci.7 604. 66 . Twyman and 19.Mathers (1987) Synapse1 96. 20 . Barker et al (1982) J.Physiol. Macdonald(1992) J.Physiol.456 215.67 . Adkins et al (2001) 322365.21 .Barker and Mathers (1981) Science 212 358. J.Biol.Chem.276 38934.68 .Smith et al (1998) Nature 392 926. 22.Hamillet al (1981) Pflugers Arch.391 85. 23 . Bormann et al 69.Smithet al (1998) J.Neurosci.18 5275. 70 . Franks and Lieb (1987) J.Physiol.385 243.24 .Macdonald et al (1989) J.Physiol. 410 (1998) Toxicol.Lett.100 1.71 .Belelli et al (1997) Proc.Natl.Acad.Sci. 479.25 .Twymanet al (1990) J.Physiol.423 193. 26 . Bowery et al USA94 11031.72 .Thompson and Wafford (2001) Curr.Opin. (1976) Br.J.Pharmacol.57 435.27 .Twyman et al (1992) J.Physiol. Pharmacol.138 78.73 .Enz and Cutting (1998) Vision Res. 1431. 44597.28 .Richards et al (1987) J.Neurosci. 7 1866. 74.Enz and Cutting (1999) Eur.J.Neurosci.11 41. 75 . Bormann 29.Stephenson and Barnard (1986) in Benzodiazepine/GABA (2000) TiPS21 16.76 .Bowery and Enna (2000) J.Pharmacol.Exp. chloride channels: structural and functional properties, p261, Alan R. Ther.292 2.77 .Kaupmannet al ( 1997) Nature 368 239. 78 . Jones Liss.30 .Schofieldet al (1987) Nature328 221. 31 . Hevers and et al(1998) Nature396 674.79 .Kaupmann et al (1998) Nature 396 Luddens(1998) Mol.Neurobiol.18 35.32 . Nayeem et al (1994) 683.80 .Whiteet al (1998) Nature396 679. 81 . Kuner et al (1999) J.Neurochem.62 815.33 .Whiting et al (1996) Int.Rev.Neurobiol. 38 Science283 74.82 .Billintonet al (2001) TiNS 24 277. 83 . Ng et al 95.34 .Wisdenet al (1992) J.Neurosci.12 1040. 35 . Farrar et al (2001) Mol.Pharmacol.59 144.84 .Galvez et al (1999) J.Biol.Chem. (1999) J.Biol.Chem.274 10100.36 .Amin and Weiss (1993) Nature 27413362.85 .Galvez et al (2000) Mol.Pharmacol. 57 419. 366565.37 .Smith and Olsen (1995) TiPS 16 162. 38 . Brejc et al 86.Murata et al (1996) Bioorg.Med.Chem.Lett.6 2071. (2001) Nature411 269.39 .Squires and Braestrup (1976) Nature 87.Raggozinoet al (1996) Mol.Pharmacol.50 1024. 88 . Schmidt et 266732.40 .Mohler and Okada (1976) 267 65. 41 . Sieghart (1995) al(2001) J.Neurosci.21 691.89 .Quirk et al (1996) Pharmacol.Rev.47 181.42 .Duncalfe et al (1996) J.Biol.Chem. 271 Neuropharmacology35 1331.90 .Hadingham et al (1993) Mol. 9209.43 .Wieland et al (1992) J.Biol.Chem.267 1426. Pharmacol.43 970.91 .Zhang et al (2001) TiPS 22 121. 44.Squires et al (1979) Pharmacol.Biochem.Behav.10 825.

7 GABA Receptor Compounds available from Tocris

GABAA Receptor Compounds Agonists 1088 CGP 54626 ...... Potent, selective GABAB 0344 GABA ...... Endogenous agonist antagonist 0235 Isoguvacine ...... Specific GABAA agonist R1088[3 H]-CGP 54626b ...... Radiolabelled form of (1088) 0289 Muscimol ...... Potent GABAA agonist 1248 CGP 55845 ...... Potent, selective GABAB 0381 Quisqualamine ...... Weak GABAA agonist antagonist 0181 TACA...... GABAA agonist. Also GABA-T 0245 2-Hydroxysaclofen...... Selective GABAB antagonist, substrate and GABAuptake inhibitor more potent than 0807 THIP ...... GABAA agonist (0246) 0180 ZAPA ...... Agonist at ‘low affinity’ GABAA 0178 Phaclofen ...... Weak, selective GABAB receptor. More potent than GABA/ antagonist muscimol 0246 Saclofen ...... Selective GABAB antagonist Antagonists 0984 SCH 50911...... Selective, competitive, orally 0130 (+)-Bicuculline ...... Potent GABAA antagonist active GABAB antagonist 0109 (-)-Bicuculline ...... Water soluble GABAA antagonist Other methobromide 1513 CGP 7930 ...... Positive modulator at GABAB 0131 (-)-Bicuculline ...... Water soluble GABAA antagonist receptors methochloride 1514 CGP 13501 ...... Positive modulator at GABAB 1128 Picrotoxin ...... GABAA receptors 1262 SR 95531 ...... Selective, competitive GABAA receptor antagonist GABAC Receptor Compounds Other Agonists 0311 CHEB ...... Convulsant 0179 CACA ...... Partial GABAC agonist 0881 Chlormethiazole ...... Potentiates GABAA receptor function 0344 GABA ...... Endogenous agonist 0505 Dihydroergotoxine ...... Binds with high affinity to GABAA 0815 -4-acetic acid....Partial GABAC agonist – receptor Cl channel 0289 Muscimol ...... Partial GABAC agonist 1471 Etomidate ...... GABA mimetic and GABA 0181 TACA...... GABAC agonist modulatory agent Antagonists 1295 ...... Subtype-selective GABAA R1301[3 H]-P4MPAb ...... GABA antagonist C 0379 SKF 97541 ...... Potent GABAB agonist. Also 0830 Primidone ...... Potentiates GABAA receptor function GABAC antagonist Benzodiazepines and Related Compounds 0807 THIP ...... GABAC antagonist 1040 TPMPA *...... Selective GABAC antagonist 0865 1-Amino-5-bromouracil....Agonist at benzodiazepine-GABA A 0180 ZAPA ...... GABAC antagonist site 0405b -CCB ...... Inverse agonist, putative Inhibitory Amino Acid Uptake Inhibitors endogenous ligand 0206b -Alanine...... Distinguishes GABA 0612b -CCP ...... Inverse agonist transporters 0456 ...... Skeletal muscle relaxant 1296 CI 966...... Selective inhibitor of GAT1 0554 FG-7142 ...... Inverse agonist 0234 Guvacine ...... Specific GABA uptake inhibitor 0658 FGIN-1-27 ...... Potent, specific ligand for 0236 (±)-Nipecotic acid ...... GABA uptake inhibitor mitochondrial DBI receptor 0768 Riluzole ...... GABA uptake inhibitor. Also 0659 FGIN-1-43 ...... Potent, specific ligand for glutamate release inhibitor mitochondrial DBI receptor 1081 SKF 89976A...... Potent GABA uptake inhibitor. 1328 Flumazenil...... Benzodiazepine antagonist Penetrates blood brain barrier 0770 GBLD 345 ...... High affinity agonist 0181 TACA...... GABA uptake inhibitor. Also 1327 L-655,708 ...... Selective fora 5-containing GABAA GABAA agonist and substrate receptors for GABA-T R1327[3 H]-L-655,708b ...... Radiolabelled form of (1327) 0670 PK-11195...... Antagonist at peripheral BDZ Miscellaneous GABA/ Compounds receptors 0806 Gabapentin...... Anticonvulsant. Increases brain 0655 Zolpidem * ...... Benzodiazepine agonist GABA 1094 Zopiclone...... Benzodiazepine agonist 0538trans -4-Hydroxycrotonic ..GHB receptor ligand acid GABAB Receptor Compounds 1260 ...... Modulates glutamate/GABA- Agonists activated CI- channels 0417 (RS )-Baclofen...... Selective GABAB agonist 0386 3-Methyl-GABA ...... Activator of GABA amino- 0796 (R )-Baclofen ...... Active enantiomer transferase 0344 GABA ...... Endogenous agonist 0780 NCS-382 ...... Antagonist ofg hydroxybutyric 0379 SKF 97541 ...... Extremely potent GABAB agonist acid Antagonists 0939 Propofol...... Potentiates GABAA receptor- mediated inhibition and inhibits 1245 CGP 35348 ...... Brain penetrant, selective GABAB antagonist NMDA receptor-mediated excitation 1247 CGP 46381 ...... Brain penetrant, selective GABAB antagonist 0808 ...... GABA-T inhibitor 1246 CGP 52432 ...... Potent, selective GABAB antagonist *Local regulations may restrict the sale of these products in certain territories. Please consult your local Tocris Cookson office or distributor for further details.

GABA Receptors, Tocris Reviews No. 20, March 2002

©2002 Tocris Cookson Published and distributed by Tocris Cookson, Bristol, UK

Editors: Samantha Manley, Ph.D., Natalie Barker, B.Sc. Design and Production: Jane Champness

GABA[Rev](0302)

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