GABA RECEPTORS Ian L Martin and Susan M J Dunn receptors. The receptor became known as GABAC . The DNAs that encode these receptor Ian Martin is Professor of Pharmacology 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 ion channel 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 muscimol, isoguvacine, 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 radioligands. 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 bicuculline.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 baclofen, 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 Gabapentin (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-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 picrotoxin and by a number of GABAA receptors have been purified from the bicyclophosphates.26 Penicillin 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 acetylcholine receptor. 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 Phaclofen (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 benzodiazepine (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.