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.