Tocris Scientific Review Series Tocri-lu-2945 GABA Receptors Ian L. Martin, Norman G. Bowery Historical Perspective and Susan M.J. Dunn GABA is the major inhibitory amino acid transmitter of the Ian Martin is Professor of Pharmacology in the School of Life and mammalian central nervous system (CNS). Essentially all neurons Health Sciences, Aston University, Birmingham, UK. Norman in the brain respond to GABA and perhaps 20% use it as their 1 Bowery is Emeritus Professor of Pharmacology, University of primary transmitter. Early electrophysiological studies, carried Birmingham, UK. Susan Dunn is Professor and Chair at the out using iontophoretic application of GABA to CNS neuronal Department of Pharmacology, Faculty of Medicine and Dentistry, preparations, showed it to produce inhibitory hyperpolarizing 2 University of Alberta, Canada. All three authors share common responses that were blocked competitively by the alkaloid 3 interests in GABAergic transmission. E-mail: sdunn@pmcol. bicuculline. However, in the late 1970s, Bowery and his ualberta.ca colleagues, who were attempting to identify GABA receptors on peripheral nerve terminals, noted that GABA application reduced the evoked release of noradrenalin in the rat heart and that this Contents effect was not blocked by bicuculline. This action of GABA was Introduction ............................................................................................. 1 mimicked, however, by baclofen (Figure 1), a compound that was unable to produce rapid hyperpolarizing responses in central The GABAA Receptor ...........................................................................1–9 neurons. This newly identified receptor was named GABAB to Distribution and Function ................................................................. 1 differentiate it from the more familiar receptor type which became Receptor Diversity ............................................................................. 2 4,5 known as GABAA. Another bicuculline-insensitive receptor was Structure and Function ..................................................................... 3 first identified using the conformationally restricted GABA analog, 6,7 CACA (Figure 1). This receptor, previously termed GABAC, has Modulators of GABAA Receptor Function ......................................... 6 now been subsumed into the GABAA receptor class, on the Receptors Previously Classified as GABA ....................................... 9 C recommendation of the IUPHAR Nomenclature Committee.8 The GABAB Receptor ......................................................................10–12 Distribution and Function ...............................................................10 The GABAA Receptor Molecular Characterization ............................................................10 Distribution and Function Clinical Potential ..............................................................................11 GABAA receptors are widely but differentially distributed within Conclusions ...........................................................................................12 the CNS.9 These receptors can be activated by a number of GABA 10 References .....................................................................................12–13 isosteres, including muscimol and isoguvacine (Figure 1). After radiolabeling, some of these ligands proved valuable in the early GABA Receptor Compounds .........................................................14–15 delineation of receptor distribution. Functionally, receptor Figure 1 | Structures of selected GABA receptor agonists Cl O H OH H2N O H N OH H2N P 2 O HO H H O H2N OH H CACA (1S,2R)-(+)-2-(Aminomethyl)- 3-Aminopropyl (R)-Baclofen cis-Aminocrotonic acid cyclopropane-1-carboxylate phosphinic acid (Cat. No. 0796) CO2H OH OH O H2NP H2N CO2H H2N N Me OH N HN N O O H GABA Isoguvacine Muscimol SKF 97541 THIP (Cat. No. 0344) (Cat. No. 0235) (Cat. No. 0289) (Cat. No. 0379) (Cat. No. 0807) (Bold text denotes compounds available from Tocris at time of publication) tocris.com | 1 Tocris Scientific Review Series activation results in an increased membrane chloride SR 95531 hydrobromide, a selective, competitive conductance,11,12 usually causing an influx of Cl– and membrane GABA antagonist hyperpolarization. In general, concentration-response curves A exhibit positive cooperativity, which is consistent with the Cat. No. 1262 presence of at least two agonist binding sites on each receptor NH 13–15 OH molecule. On continued exposure to high agonist N concentrations, the agonist-induced current decreases as a N O consequence of receptor desensitization.16–18 Biophysical characterization of the receptor, carried out initially using noise analysis of neurons in primary culture, provided the first estimates of mean single channel conductance and average channel open OMe 19 times, the latter of which varied with the nature of the activating SR 95531 is a selective, competitive GABA receptor 20 21 A agonist. Development of single channel recording techniques antagonist that displaces [3H]-GABA from rat brain membranes provided further detail on the nature of single channel events with a Ki value of 150 nM. Unlike bicuculline, SR 95531 with the demonstration of multiple single channel conductances: selectively antagonizes GABA-induced Cl– currents with little 44, 30, 19 and 12pS,22 the 30pS conductance being the most action on pentobarbitone-induced currents. The compound prevalent. Both channel opening times and opening frequency also acts as a low affinity glycine receptor antagonist. are dependent on agonist concentration and the competitive Heaulme et al. (1986) Brain Res. 384 224. Uchida et al. (1996) Eur.J.Pharmacol. 307 89. Beato et al. (2007) J.Physiol. 580 171. antagonist, bicuculline, reduces the conductance by modulating both of these parameters.23,24 Other competitive antagonists include the pyridazinyl GABA derivative, SR 95531 (Figure 2). The also facilitated the cloning of the first two GABAA receptor subunit receptor can also be blocked non-competitively by picrotoxin and isoforms.28,29 a number of bicyclophosphates.25 In addition, penicillin decreases channel open probability in a manner that is compatible with Subsequent molecular studies revealed a multiplicity of protein open channel block.26 subunits that have now been divided into seven classes, based on the extent of similarities in their deduced amino acid Receptor Diversity sequences. Within these classes there are further subdivisions into subunit isoforms, some of which exhibit alternate splice Purification of the bovine brain receptor in the early 1980s variants. In man, six α-, three β-, three γ- and three ρ- subunit revealed two major subunits of the GABA receptor, which were A isoforms are presently known, together with single representatives α β named and . Elucidation of partial amino acid sequences of of the δ, ε, π and θ classes. Within a single subunit class, the these subunits allowed subunit-specific monoclonal antibodies sequence homology is about 70% but between classes this falls to be raised, thus providing the opportunity to explore the fine to around 30%. Additional isoforms of some of these classes are 27 anatomical detail of receptor distribution. The sequence data known in other species.30 In the earlier receptor nomenclature, Figure 2 | Structures of selected GABA receptor agonists O OEt NMe O H H HO O N P OEt HN P H O H2N P OEt OO H OH O OH Cl Me OEt O Cl Cl O O Cl (+)-Bicuculline CGP 35348 CGP 52432 CGP 55845 (Cat. No. 0130) (Cat. No. 1245) (Cat. No. 1246) (Cat. No. 1248) NH OH N Me N O SO2OH O H HN P OH CO2H Cl O Me NH2 N Me H OMe TPMPA Saclofen SCH 50911 SR 95531 (1,2,5,6-tetrahydropyridin- (Cat. No. 0246) (Cat. No. 0984) (Cat. No. 1262) 4-yl)methylphosphinic acid (Cat. No. 1040) (Bold text denotes compounds available from Tocris at time of publication) 2 | tocris.com GABA Receptors the three ρ-subunits were considered to define the GABA C (–)-Bicuculline methochloride, a water-soluble GABA receptor.8 A antagonist Deduced amino acid sequences from each of the subunits reveal Cat. No. 0131 homologies and a common structural organization which places O them firmly within the so-called Cys-loop ligand-gated ion channel N+Me O 2 (LGIC) family. These receptors are pentamers of homologous H H subunits that assemble to form a central ion channel traversing O the cell membrane. The archetypal member of the family is the O O peripheral nicotinic acetylcholine receptor (nAChR) with other O members of the family including glycine and 5-HT3 receptors. Each subunit has a long amino terminal domain of more than (–)-Bicuculline methochloride is a water soluble and more 200 amino acids which carries the signature cys-cys loop. This stable salt of (+)-bicuculline (Cat. No. 0130) that acts as a com- extracellular domain is followed by four hydrophobic segments, petitive GABAA receptor antagonist. The compound blocks – each of which is about 20 amino acids long. These four segments, inhibitory hyperpolarizing responses and reduces Cl conduc- tance by modulating channel opening time and frequency. termed TM1–TM4, were predicted to form transmembrane Kemp et al. (1986) Br.J.Pharmacol. 87 677. MacDonald et al. (1989) J.Physiol. domains with TM2 contributing to formation of the ion channel 410 479. Seutin and Johnson (1999) TiPS 20 268. lining. Between TM3 and TM4 there is a large intracellular loop, which is the most divergent part of the sequence within the GABAA receptor subfamily. characteristics of individual