Stable expression of mammalian type A gamma-aminobutyric acid receptors in mouse cells: demonstration of functional assembly of benzodiazepine-responsive sites.
- 15 July 1992
- journal article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 89 (14) , 6378-6382
- https://doi.org/10.1073/pnas.89.14.6378
Abstract
The differential sensitivity of type A gamma-aminobutyric acid (GABAA) receptors to benzodiazepine ligands seen in the mammalian 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 (alpha 1-6, beta 1-3, gamma 1-3, and delta) encoded by discrete genes. The characteristics of a given subtype can be determined by the coexpression of cloned cDNAs encoding the subunits of interest. 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 alpha 1 beta 1 gamma 2L GABAA receptor subtype. Using both immunoprecipitation by subunit-specific antisera and gel-exclusion chromatography, we have shown that the alpha 1, beta 1, and gamma 2L subunits coassemble to form receptor macromolecules that are of the same size as native GABAA receptors. Additionally, the recombinant receptors have the same benzodiazepine pharmacology as native alpha 1-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 of GABAA receptors and in the development of subtype-specific drugs.Keywords
This publication has 28 references indexed in Scilit:
- Structural and pharmacological characterization of the major brain nicotinic acetylcholine receptor subtype stably expressed in mouse fibroblasts.1991
- GABAA receptor subtypes immunopurified from rat brain with α subunit-specific antibodies have unique pharmacological propertiesNeuron, 1991
- Cloning, pharmacological characteristics and expression pattern of the rat GABAA receptor α4 subunitFEBS Letters, 1991
- N‐Deglycosylation and immunological identification indicates the existence of β‐subunit isoforms of the rat GABAA receptorFEBS Letters, 1991
- A novel γ subunit of the GABAA receptor identified using the polymerase chain reactionFEBS Letters, 1991
- Effects of (+)-HA-966 and 7-chlorokynurenic acid on the kinetics of N-methyl-D-aspartate receptor agonist responses in rat cultured cortical neurons.1991
- Another mechanism for creating diversity in gamma-aminobutyrate type A receptors: RNA splicing directs expression of two forms of gamma 2 phosphorylation site.Proceedings of the National Academy of Sciences, 1990
- Functional properties of recombinant rat GABAA receptors depend upon subunit compositionNeuron, 1990
- Calcium phosphate-mediated gene transfer: a highly efficient transfection system for stably transforming cells with plasmid DNA.1988
- Some properties of brain specific benzodiazepine receptors: New evidence for multiple receptorsPharmacology Biochemistry and Behavior, 1979