␥-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian brain. The GABA receptor type C (GABA C ) is a ligand-gated ion channel with pharmacological properties distinct from the GABA A receptor. To date, only three binding domains in the recombinant 1 GABA C receptor have been recognized among six potential regions. In this report, using the substituted cysteine accessibility method, we scanned three potential regions previously unexplored in the 1 GABA C receptor, corresponding to the binding loops A, E, and F in the structural model for ligand-gated ion channels. The cysteine accessibility scanning and agonist/antagonist protection tests have resulted in the identification of residues in loops A and E, but not F, involved in forming the GABA C receptor agonist binding pocket. Three of these newly identified residues are in a novel region corresponding to the extended stretch of loop E. In addition, the cysteine accessibility pattern suggests that part of loop A and part of loop E have a ␤-strand structure, whereas loop F is a random coil. Finally, when all of the identified ligand binding residues are mapped onto a three-dimensional homology model of the amino-terminal domain of the 1 GABA C receptor, they are facing toward the putative binding pocket. Combined with previous findings, a complete model of the GABA C receptor binding pocket was proposed and discussed in comparison with the GABA A receptor binding pocket. GABA A1 and GABA C receptors are both GABA-gated chloride channels but with very different pharmacological properties. In fact, that is the basis for their classification. Although both types of receptors can be activated by GABA, the GABA A receptor can be specifically antagonized by bicuculline. In contrast, the GABA C receptor is insensitive to bicuculline but can be selectively antagonized by 1,2,5,6-tetrahydropyridine-4-yl)-methylphosphinic acid (1). The functional properties of these two types of GABA receptors are also different. For example, the GABA C receptor has higher GABA sensitivity but slower activation and deactivation kinetics than the GABA A receptor (2). The GABA C receptor also shows almost no desensitization (2), whereas the GABA A receptor exhibits strong desensitization (3). Although the functional differences could arise from differences in the structural architecture of the GABA binding pockets, distinct antagonist profiles of GABA A and GABA C receptors indicate that their agonist/antagonist binding pockets are not the same. Studies over the last 15 years have shaped a relatively complete model for the GABA A receptor (4 -9). In contrast, structural information on the GABA C receptor agonist binding pocket is far from complete. Some candidate binding residues in several potential "binding loops" are still undefined.GABA-gated ion channels belong to the ligand-gated ion channel family, which also includes nicotinic acetylcholine receptors, serotonin-3 receptors, and glycine receptors. The study of nicotinic receptors in the past two decades h...

Background and purpose: This study investigated the a 1 b 2 d isoform of the GABA A receptor that is presumably expressed in the forebrain. The functional and pharmacological properties of this receptor combination are largely unknown. Experimental approach: We expressed a 1 b 2 d GABA A receptors in Xenopus laevis oocytes. GABA-activated currents, in the presence and absence of modulators, were recorded using the two-electrode voltage clamp technique. Key results: The a 1 b 2 d isoform of the GABA A receptor exhibited an extremely small GABA-mediated current. Tracazolate increased the current amplitude evoked by a half-maximal concentration (EC 50 ) of GABA by 59-fold. The maximum current was increased 23-fold in the presence of a saturating GABA concentration. Concomitant with the increase in the maximum, was a 4-fold decrease in the EC 50 . Finally, a mutation in the second transmembrane domain of the d subunit that increases receptor efficacy (L286S), eliminated the increase in the maximum GABA-activated current. The endogenous neurosteroid, tetrahydrodeoxycorticosterone (THDOC), also decreased the EC 50 and increased the maximum current amplitude, although to a lesser degree than that of tracazolate. Conclusions and implications: Taken all together, these findings indicate that the small GABA-mediated currents in the absence of the modulator are due to a low efficacy for activation. In the absence of modulators, a 1 b 2 d GABA receptors would be effectively silent and therefore contribute little to inhibition in the CNS. In the presence of tracazolate or endogenous neurosteroids however, this particular receptor isoform could exert a profound inhibitory influence on neuronal activity.

Structure-function studies of the Cys loop family of ionotropic neurotransmitter receptors (GABA, nACh, 5-HT(3), and glycine receptors) have resulted in a six-loop (A-F) model of the agonist-binding site. Key amino acids have been identified in these loops that associate with, and stabilize, bound ligand. The next step is to identify the structural rearrangements that couple agonist binding to channel opening. Loop F has been proposed to move upon receptor activation, although it is not known whether this movement is along the conformational pathway for channel opening. We test this hypothesis in the GABA receptor using simultaneous electrophysiology and site-directed fluorescence spectroscopy. The latter method reveals structural rearrangements by reporting changes in hydrophobicity around an environmentally sensitive fluorophore attached to defined positions of loop F. Using a series of ligands that span the range from full activation to full antagonism, we show there is no correlation between the rearrangements in loop F and channel opening. Based on these data and agonist docking simulations into a structural model of the GABA binding site, we propose that loop F is not along the pathway for channel opening, but rather is a component of the structural machinery that locks ligand into the agonist-binding site.

αβδ-containing GABAA receptors are 1) localized to extra- and peri-synaptic membranes, 2) exhibit a high sensitivity to GABA, 3) show little desensitization, and 4) are believed to be one of the primary mediators of tonic inhibition in the central nervous system. This type of signaling appears to play a key role in controlling cell excitability. This review article briefly summarizes recent knowledge on tonic GABA-mediated inhibition. We will also consider the mechanism of action of many clinically important drugs such as anxiolytics, anticonvulsants, and sedative/hypnotics and their effects on δ-containing GABA receptor activation. We will conclude that αβδ-containing GABAA receptors exhibit a relatively low efficacy that can be potentiated by endogenous modulators and anxiolytic agents. This scenario enables these particular GABA receptor combinations, upon neurosteroid exposure for example, to impart a profound effect on excitability in the central nervous system.

Picrotoxin, a potent antagonist of the inhibitory central nervous system GABA A and glycine receptors, is believed to interact with residues that line the central ion pore. These pore-lining residues are in the second transmembrane domain (TM2) of each of the five constituent subunits. One of these amino acids, a threonine at the 6 location, when mutated to phenylalanine, abolishes picrotoxin sensitivity. It has been suggested that this threonine, via hydrogen bonding, directly interacts with the picrotoxin molecule. We previously demonstrated that this mutation, in the α, β or γ subunit, can impart picrotoxin resistance to the GABA receptor. Since the functional pentameric GABA receptor contains two α subunits, two β subunits and one γ subunit, it is not clear how many α and β subunits must carry this mutation to impart the resistant phenotype. In this study, by coexpression of mutant α or β subunits with their wild-type counterparts in various defined ratios, we demonstrate that any single subunit carrying the 6 mutation imparts picrotoxin resistance. Implications of this finding in terms of the mechanism of antagonism are considered.