Stable expression of mammalian type A gamma-aminobutyric acid receptors in mouse cells: demonstration of functional assembly of benzodiazepine-responsive sites.

Abstract

The differential sensitivity of type A -aminobutyric acid (GABAA) receptors to benzodiazepine lgands seen in the malian nervous system is thought to be generated by the existence of a number of different receptor subtypes, assembled from a range of closely related subunits (a1-6, P1-3, V1-3, and 6) encoded by discrete genes. The characteristics of a given subtype can be determined by the coexpression ofcloned cDNAs encoding the subunits ofinterest. Two transient expression systems have so far been employed in the study of the ligand-binding characteristics and chloride channel properties of such GABAA receptors-Xenopus oocytes and transfected mammalian cells. Here we report on the use of a steroid-inducible promoter expression system for the production of a permanently transfected clonal cell line expressing the aj.8Vy2L GABAA receptor subtype. Using both immunoprecipitation by subunit-specific antisera and gelexclusion chromatography, we have shown that the ai, 13i, and 'Y2L subunits coassemble to form receptor macromolecules that are of the same size as native GABAA receptors. Additionally, the recombinant receptors have the same bendiazepine pharmacology as native a1-containing GABAA receptors and function as GABA-gated chloride channels. Such cell lines expressing individual GABAA receptor subtypes will prove important tools in the study of the structure, function, and pharmacology ofGABAA receptors and in the development of subtype-specific drugs.In the mammalian brain, type A y-aminobutyric acid (GABAA) receptors are the major mediators of inhibitory neurotransmission. They constitute GABA-gated chloride channels, whose activity may be allosterically modulated by a number of drugs, including the barbiturates and benzodiazepines (BZs) (1). Heterogeneity in the responses of the mammalian receptors to BZ-type ligands has been attributed to the differential assembly of GABAA receptor subtypes from a family of subunits (al16, 11-3, y1-3, and 8) that have been identified by recombinant DNA approaches (2-14).As individual GABAA receptor subtypes are not known to be expressed in any transformed cell lines, it has proved necessary to employ other means for their in vitro analysis. Transient expression of various subunit combinations in Xenopus oocytes (2-7) and human embryonic kidney 293 cells (7)(8)(9)(10)(11)(15)(16)(17) have been used to investigate their functional and ligand-binding properties. These approaches have suggested that, while there is a requirement for an a subunit, a 13 subunit, and the Y2 subunit in order to allow BZ binding (8), the relative affinities for various ligands, as classically defined by BZ-1 or BZ-2 pharmacology (18) (2) were then subcloned into the modified pMSGneo. The bovine 'Y2L subunit cDNA (12) was modified for transfection by addition of 5' untranslated-region sequence derived from the bovine a, clone. Briefly, oligonucleotides 5'-GCGGAGC-

Identification of Residues within GABAAReceptor α Subunits That Mediate Specific Assembly with Receptor β Subunits

“…Together, these results demonstrate that Q67 and S68 within the ␣1 subunit are critical in mediating assembly with ␤3 to form 9S complexes, representing functional cell surface receptors (Mamalaki et al, 1987(Mamalaki et al, , 1989Hadingham et al, 1992;Gorrie et al, 1997;Tretter et al, 1997). (9E10) ␣1 (HY) subunits appear to oligomerize less efficiently with (FL AG) ␤3 compared to (9E10) ␣1 and are rapidly degraded as previously described for unassembled wild-type ␣1 subunits (Gorrie et al, 1997).…”

“…Quantitation of the gradients revealed that both proteins exhibited 9 S sedimentation coefficients, as previously described for functional GABA A receptors composed of ␣␤ or ␣␤␥ subunits (Fig. 6 A,B; Mamalaki et al, 1987Mamalaki et al, , 1989Hadingham et al, 1992;Gorrie et al, 1997;Tretter et al, 1997). In contrast, unassembled ␣ or ␤ subunits have 5 S sedimentation coefficients (Gorrie et al, 1997;Tretter et al, 1997).…”

Ethanol potentiation of GABAA receptors requires phosphorylation of the alternatively spliced variant of the γ2 subunit

“…The bovine r,l, fll,72L and 72S cDNAs have been described previouqy [l, 12,24]. cRNA was prepared from 0tl and fll ¢DNAs a~ prev.…”

Comparative Cue Generalization Profiles of L-838, 417, SL651498, Zolpidem, CL218,872, Ocinaplon, Bretazenil, Zopiclone, and Various Benzodiazepines in Chlordiazepoxide and Zolpidem Drug Discrimination

“…a) One group included compounds with selective affinity, including the imidazopyridine zolpidem and triazolopyridazine CL218,872, ligands that selectively bind to GABA A -␣ 1 receptors. These compounds selectively potentiate the effect of GABA at GABA A -␣1 receptors, although this is concentration-dependent (i.e., they only show 10 -20-fold affinity selectivity for GABA A -␣ 1 receptors over GABA A -␣ 2 or -␣ 3 receptors), despite Ͼ1000-fold selectivity over GABA A -␣ 5 receptors (Hadingham et al, 1992;Faure-Halley et al, 1993;Sieghart, 1995). To add to the complexity, zolpidem has highintrinsic efficacy at GABA A -␣ 1 receptors, whereas CL218,872 displays reduced efficacy at this receptor; i.e., CL218,872 is a partial agonist at GABA A -␣ 1 receptors.…”