No abstract

Fast inhibitory neurotransmission in the mammalian CNS is mediated primarily by the neurotransmitter gamma-aminobutyric acid (GABA), which, upon binding to its receptor, leads to opening of the intrinsic ion channel, allowing chloride to enter the cell. Over the past 10 years it has become clear that a family of GABA-A receptor subtypes exists, generated through the coassembly of polypeptides selected from alpha 1-alpha 6, beta 1-beta 3, gamma 1-gamma 3, delta, epsilon, and pie to form what is most likely a pentomeric macromolecule. The gene transcripts, and indeed the polypeptides, show distinct patterns of temporal and spatial expression, such that the GABA-A receptor subtypes have a defined localization that presumably reflects their physiological role. A picture is beginning to emerge of the properties conferred to receptor subtypes by the different subunits; these include different functional properties, differential modulation by protein kinases, and the targeting to different membrane compartments. These properties presumably underlie the different physiological roles of the various receptor subtypes. Recently we have identified a further member of the GABA-A receptor gene family, which we have termed theta, which appears to be most closely related to the beta subunits. The structure, function, and distribution of theta-containing receptors, and receptors containing the recently reported epsilon subunit, are described.

The GABA A receptor subtypes responsible for the anxiolytic effects of nonselective benzodiazepines (BZs) such as chlordiazepoxide (CDP) and diazepam remain controversial. Hence, molecular genetic data suggest that ␣2-rather than ␣3-containing GABA A receptors are responsible for the anxiolytic effects of diazepam, whereas the anxiogenic effects of an ␣3-selective inverse agonist suggest that an agonist selective for this subtype should be anxiolytic. We have extended this latter pharmacological approach to identify a compound, 4,2Ј-difluoro-5Ј-[8-fluoro-7-(1-hydroxy-1-methylethyl)imidazo[1,2-á]pyridin-3-yl]biphenyl-2-carbonitrile (TP003), that is an ␣3 subtype selective agonist that produced a robust anxiolytic-like effect in both rodent and non-human primate behavioral models of anxiety. Moreover, in mice containing a point mutation that renders ␣2-containing receptors BZ insensitive (␣2H101R mice), TP003 as well as the nonselective agonist CDP retained efficacy in a stress-induced hyperthermia model. Together, these data show that potentiation of ␣3-containing GABA A receptors is sufficient to produce the anxiolytic effects of BZs and that ␣2 potentiation may not be necessary.

We report the isolation and characterization of a cDNA encoding a novel member of the GABA receptor gene family, ⑀. This polypeptide is 506 amino acids in length and exhibits its greatest amino acid sequence identity with the GABA A receptor ␥3 subunit (47%), although this degree of homology is not sufficient for it to be classified as a fourth ␥ subunit. The ⑀ subunit coassembles with GABA A receptor ␣ and ␤ subunits in Xenopus laevis oocytes and transfected mammalian cells to form functional GABA-gated channels. ␣1␤1⑀ GABA A receptors, like ␣1␤1␥2s receptors, are modulated by pentobarbital and the steroid 5␣-pregnan-3␣-ol-20-one but, unlike ␣1␤1␥2s receptors, are insensitive to flunitrazepam. Additionally, ␣1␤1⑀ receptors exhibit rapid desensitization kinetics, as compared with ␣1␤1 or ␣1␤1␥2s. Northern analysis demonstrates widespread expression of a large ⑀ subunit transcript in a variety of nonneuronal tissues and expression of a smaller transcript in brain and spinal cord. Sequence analysis demonstrated that the large transcript contained an unspliced intron, whereas the small transcript represents the mature mRNA, suggesting regulation of expression of the ⑀ subunit via neuronally restricted RNA splicing. In situ hybridization and immunocytochemistry reveal a pattern of expression in the brain restricted primarily to the hypothalamus, suggesting a role in neuroendocrine regulation, and also to subfields of the hippocampus, suggesting a role in the modulation of long term potentiation and memory.

Specimens from the human male and female external urethral sphincter and the periurethral levator ani muscle have been examined using histochemical and electron microscopic techniques. In both sexes the external sphincter consists of a single population of type I (slow twitch) fibres with a mean diameter of 17.47 +/- 0.7 micrometers in the absence of muscle spindles. In contrast, the periurethral levator ani possesses muscle spindles and the constituent fibres form a heterogeneous population of type I and type II (fast twitch) fibres, with mean diameters of 45.5 +/- 0.8 micrometer and 59.5 +/- 3.4 micrometers respectively. These findings indicate that the external urethral sphincter is functionally adapted to maintain tone over prolonged periods and may be of considerable importance in producing active urethral closure during continence. The anatomical location and fibre characteristics of the levator ani muscle suggest that these fibres actively assist in urethral closure, particularly during events which cause elevation of intra-abdominal pressure. In view of the differences in fibre characteristics between the external urethral sphincter and the levator ani, EMG activity recorded from a single site in the levator ani may not be representative of the functional status either of other levator ani muscle fibres or of the external urethral sphincter.

1 Human GABAA receptors containing different and subunits with a y2s subunit were expressed in Xenopus oocytes and the effects of pentobarbitone on these subunit combinations were examined by electrophysiological recording of GABA currents with the two-electrode voltage-clamp method. 2 Pentobarbitone has previously been shown to have three actions on GABAA receptors: a potentiation of GABA responses, a direct activation of GABAA receptors and, at high concentrations, a block of the GABA chloride channel. In this study pentobarbitone activity consisted of the above mentioned three components on all the subunit combinations tested. However, the affinities and efficacies varied with receptor subtype. 3 Potentiation of GABA by pentobarbitone occurred over the same concentration-range for all the subunits with affinities in the range of 20-35 tiM. The degree of potentiation obtained, however, varied from 236% of GABA EC20 on al/#2y2s to 536% on a6fl2y2s.4 Examination of the direct effect of pentobarbitone revealed that the type of a subunit present determines both the degree of affinity and efficacy obtained. Receptors containing an cc6 subunit produced maximum direct responses to pentobarbitone larger than that obtainable with maximum GABA (150% to 170% of maximum GABA). The maximum direct pentobarbitone response obtainable with other a subunits ranged between 45% of maximum GABA for ac5f2y2s to 82% for a2/32y2s. The affinity of the direct action of pentobarbitone on a6/32y2s was 58 tiM compared to affinities for the other subunits ranging from 139 pM on a2fl2y2s to 528 gM on ac5f2y2s.5 The type of subunit present did not influence the direct action of pentobarbitone to the same extent as the a subunit. There were no significant differences between affinity or efficacy on oocytes expressing a6 and y2s with /31, /32 or P3. Affinities and efficacies on oocytes expressing al and y2s with PI, /2 or /3 were significantly different with pentobarbitone having a higher affinity and efficacy on al/33y2s followed by al/32y2s and then al/3ly2s. 6 The direct effect of pentobarbitone was blocked by picrotoxin but not by competitive antagonists, such as bicuculline or SR95531, indicating that the direct agonist activity of pentobarbitone was not mediated via the GABA binding site. 7 For the first time the influence of the various a and subunits on the effects of pentobarbitone were demonstrated. The results indicate that GABAA receptors containing a6 subunits have both a higher affinity and efficacy for direct activation by pentobarbitone, and reveal that pentobarbitone binds to more than one site on the GABAA receptor, and these are dependent on receptor subunit composition.

Using human gamma-aminobutyric acid type A (GABAA) receptor subunit combinations, expressed in cell lines and Xenopus laevis oocytes, the pharmacology of a number of ligands interacting directly with the GABA recognition site has been studied in [3H]muscimol binding and electrophysiologically. The binding affinity of GABAA agonist and antagonist ligands showed small but statistically significant dependence on the subunit composition of receptors that include gamma 2 and different alpha and beta subunits. The potency of antagonist ligands was largely independent of receptor subunit composition, whereas the composition of receptors expressed in oocytes strongly influenced the EC50 value of agonists. An apparent reciprocal correlation between subunits favoring agonist binding and antagonist binding, respectively, was observed. Whereas antagonists showed comparable potencies in binding and functional studies, the potency of agonists in binding studies was generally two to three orders of magnitude higher than the agonist potencies measured electrophysiologically. 5-(4-Piperidyl)isothiazol-3-ol, which behaves as a low efficacy partial agonist at GABAA receptors in cultured cortical neurons, showed no efficacy in oocytes, but produced pure antagonist effects with a binding/functional affinity ratio between those observed for the agonists and antagonists. It is concluded that the GABAA receptor mechanisms transducing binding into physiological response, but not the binding per se, is dependent on the receptor subunit composition.

SUMMARYPharmacological analyses of ␥-aminobutyric acid A (GABA A ) receptor subtypes have suggested that both the ␣ and ␥ subunits, but not the ␤ subunit, contribute to the benzodiazepine binding site. We took advantage of the different pharmacological properties conferred by the inclusion of different ␥ subunits in the receptor macromolecule to identify amino acids ␥2Phe77 and ␥2Met130 as key determinants of the benzodiazepine binding site. ␥2Phe77 was required for high affinity binding of the benzodiazepine site ligands flumazenil, CL218,872, and methyl-␤-carboline-3-carboxylate but not flunitrazepam. This amino acid was, however, required for allosteric modulation by flunitrazepam, as well as other benzodiazepine site ligands. In contrast, ␥2Met130 was required for high affinity binding of flunitrazepam, clonazepam, and triazolam but not flumazenil, CL218,872, or methyl-␤-carboline-3-carboxylate and did not affect benzodiazepine efficacy. Introduction of the phenylalanine and methionine into the appropriate positions of ␥1 was not sufficient to confer high affinity for the benzodiazepine site ligand zolpidem. These data show that ␥2Phe77 and ␥2Met130 are necessary for high affinity binding of a number of benzodiazepine site ligands. Although most previous studies have focused on the contribution of the ␣ subunit, we demonstrated a critical role for the ␥ subunit at the benzodiazepine binding site, indicating that this modulatory site is located at the interface of these two subunits. Furthermore, ␥2Phe77 is homologous to ␣1Phe64, which has been previously shown to be a key determinant of the GABA binding site, suggesting a conservation of motifs between different ligand binding sites on the GABA A receptor.The GABA A receptor, a member of the ligand-gated ion channel family, mediates synaptic inhibition through the gating of chloride ions, resulting in hyperpolarization of the cell membrane. It is the site of action of a number of pharmacological agents, including BZs, barbiturates, and anesthetics. The hetero-oligomeric receptor is formed from the coassembly of five different subunit classes [␣, ␤, ␥, ␦ (1, 2), and ⑀ (3, 4)] in a presumed pentameric arrangement (5, 6) to yield a family of receptor subtypes. It is the heterogeneity within these subunits that provides the molecular basis for the differences in pharmacology of receptor subtypes (7).Classic BZ pharmacology is exhibited by receptors containing a ␥2 subunit in combination with an ␣ and a ␤ subunit (8). The affinity of BZ ligands for the receptor is dependent on the ␣ subunit isoform, and hence compounds such as CL218,872 and zolpidem have higher affinity for ␣1␤n␥2 (n ϭ 1, 2, or 3) receptors than for other ␣ subunit-containing receptors (9, 10), and flunitrazepam and diazepam (11, 12) have very low affinity (Ͼ10 M) for ␣4␤n␥2 and ␣6␤n␥2. Mutagenesis studies have identified two amino acids on the ␣ subunit as contributing to the BZ binding site (13,14 McKernan et al. (16) that the ␥ subunit also contributes significantly to the BZ binding site. I...