Inhibitory neurotransmission in the brain is largely mediated by GABA(A) receptors. Potentiation of GABA receptor activation through an allosteric benzodiazepine (BZ) site produces the sedative, anxiolytic, muscle relaxant, anticonvulsant and cognition-impairing effects of clinically used BZs such as diazepam. We created genetically modified mice (alpha1 H101R) with a diazepam-insensitive alpha1 subtype and a selective BZ site ligand, L-838,417, to explore GABA(A) receptor subtypes mediating specific physiological effects. These two complimentary approaches revealed that the alpha1 subtype mediated the sedative, but not the anxiolytic effects of benzodiazepines. This finding suggests ways to improve anxiolytics and to develop drugs for other neurological disorders based on their specificity for GABA(A) receptor subtypes in distinct neuronal circuits.

1 The pharmacology of the stable cell line expressing human a 4 b 3 d GABA A receptor was investigated using whole-cell patch-clamp techniques. 2 a 4 b 3 d receptors exhibited increased sensitivity to GABA when compared to a 4 b 3 g 2 receptors, with EC 50 's of 0.50 (0.46, 0.53) mM and 2.6 (2.5, 2.6) mM respectively. Additionally, the GABA partial agonists piperidine-4-sulphonate (P4S) and 4,5,6,7-tetrahydroisothiazolo-[5,4-c]pyridin-3-ol (THIP) displayed markedly higher e cacy at a 4 b 3 d receptors, indeed THIP demonstrated greater e cacy than GABA at these receptors. 3 The d subunit conferred slow desensitization to GABA, with rate constants of 4.8+0.5 s for a 4 b 3 d and 2.5+0.2 s for a 4 b 3 g 2 . However, both P4S and THIP demonstrated similar levels of desensitization on both receptor subtypes suggesting this e ect is agonist speci®c. 4 a 4 b 3 d and a 4 b 3 g 2 demonstrated equal sensitivity to inhibition by the cation zinc (2 ± 3 mM IC 50 ). However, a 4 b 3 d receptors demonstrated greater sensitivity to inhibition by lanthanum. The IC 50 for GABA antagonists SR-95531 and picrotoxin, was similar for a 4 b 3 d and a 4 b 3 g 2 . Likewise, inhibition was observed on both subtypes at high and low pH. 5 a 4 b 3 d receptors were insensitive to modulation by benzodiazepine ligands. In contrast Ro15-4513 and bretazenil potentiated GABA responses on a 4 b 3 g 2 cells, and the inverse agonist DMCM showed allosteric inhibition of a 4 b 3 g 2 receptors.6 The e cacy of neurosteroids at a 4 b 3 d receptors was greatly enhanced over that observed at a 4 b 3 g 2 receptors. The greatest e ect was observed using THDOC with 524+71.6% potentiation at a 4 b 3 d and 297.9+49.7% at a 4 b 3 g 2 receptors. Inhibition by the steroid pregnenolone sulphate however, showed no subtype selectivity. The e cacy of both pentobarbitone and propofol was slightly augmented and etomidate greatly enhanced at a 4 b 3 d receptors versus a 4 b 3 g 2 receptors. 7 We show that the a 4 b 3 d receptor has a distinct pharmacology and kinetic pro®le. With its restricted distribution within the brain and unique pharmacology this receptor may play an important role in the action of neurosteroids and anaesthetics. British Journal of Pharmacology (2002) 136, 965 ± 974

ABSTRACT␥-Aminobutyric acid type A (GABA-A) receptors are a major mediator of inhibitory neurotransmission in the mammalian central nervous system, and the site of action of a number of clinically important drugs. These receptors exist as a family of subtypes with distinct temporal and spatial patterns of expression and distinct properties that presumably underlie a precise role for each subtype. The newest member of this gene family is the subunit. The deduced polypeptide sequence is 627 amino acids long and has highest sequence identity (50.5%) with the ␤1 subunit. Within the rat striatum, this subunit coassembles with ␣2, ␤1, and ␥1, suggesting that ␥-aminobutyric acid type A receptors consisting of arrangements other than ␣␤ ؉ ␥, ␦, or do exist. Expression of ␣2␤1␥1 in transfected mammalian cells leads to the formation of receptors with a 4-fold decrease in the affinity for ␥-aminobutyric acid compared with ␣2␤1␥1. This subunit has a unique distribution, with studies so far suggesting significant expression within monoaminergic neurons of both human and monkey brain.In the mammalian central nervous system, inhibitory neurotransmission is mediated primarily by the ␥-aminobutyric acid (GABA), which acts on GABA type A (GABA-A) receptors, ligand-gated ion channels acting over a rapid time frame. 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 ␣1-␣6, ␤1-␤3, ␥1-␥3, ␦, , and to form what is most likely a pentameric macromolecule (1-6). The subunits show distinct patterns of temporal and spatial expression, so that GABA-A receptor subtypes have a defined localization presumably reflecting their physiological role (7)(8)(9)(10)(11).In this article, we report the identification and characterization of a further member of the GABA-A receptor gene family that we have termed theta (). MATERIALS AND METHODSCloning of Subunit cDNA. Full-length cDNA was cloned starting from sequence information in GenBank entry U47334. PCR was performed under standard conditions, on human whole-brain cDNA (CLONTECH) with oligonucleotide primers specific to the 5Ј and 3Ј ends of the U47334 sequence. A single PCR product of approximately 1,600 bp was obtained. The 5Ј and 3Ј ends of the coding region corresponding to full-length U47334 sequence were obtained by 5Ј and 3Ј anchored PCR using human brain Marathon cDNA (CLON-TECH). Full-length cDNA (GenBank accession no. AF144648) was generated by PCR using a primer derived from sequences surrounding the initiating methionine incorporating a consensus Kozak sequence (12), and a primer based on the 3Ј untranslated region of the anchored PCR product. The PCR product (1,958 bp) was sequenced completely on both strands by primer walking by using dye terminator chemistry and an Applied Biosystems model 373A sequencer.Epitope-tagged subunit was constructed that contained nucleotides Ϫ224 to ϩ99 (i.e., the 5Ј untranslated region, the signal peptide, 6 amino acids of the mature protein) of bovine ...

No abstract

A series of high-affinity GABA(A) agonists with good oral bioavailability in rat and dog and functional selectivity for the GABA(A)alpha2 and -alpha3 subtypes is reported. The 7-trifluoromethylimidazopyrimidine 14g and the 7-propan-2-olimidazopyrimidine 14k are anxiolytic in both conditioned and unconditioned animal models of anxiety with minimal sedation observed at full BZ binding site occupancy.