title: Effect of subunit composition on GABAA receptor complex characteristics in a baculovirus expression system.
abstract: A baculovirus expression system was used to produce functional human recombinant GABAA receptors in Sf-9 insect cells in order to study the biochemistry, pharmacology and functional characteristics of this receptor complex. We have identified and characterized various factors which influence the level of receptor expression in multiple virus infections. We have shown that the level of expression of the GABAA receptor complex varies with the levels of expression of the individual subunits. We have also shown that the assembly process has a defined timecourse, and it is dependent upon the ratio of the number of infectious virus particles (MOI ratio) of each subunit in multi-virus infections. In multiple infections, the capacity for expression of the infected cell is shared proportionally by entering virus particles and, there is a direct correlation between the amounts of subunit mRNA and levels of subunit protein expression, and the amount of ligand binding to expressed protein. Finally, reinfection of previously infected cells does not result in subsequent protein expression. Knowledge of these various factors allows us to construct recombinant GABAA receptor complexes with reproducibility and flexibility with regard to subunit composition. By co-expression of alpha, beta, and gamma subunits, both the recognition site for GABA and the allosteric (benzodiazepine) modulatory site are formed and appear to reproduce the pharmacology of endogenously expressed receptors as measured in mammalian CNS. Only a single receptor is produced irrespective of the expression levels of the subunits, showing that GABAA receptor assembly is highly regulated.
authors.affiliation: Neurogen Corporation, Dept. of Molecular Biology, Branford, CT 06405, USA.
authors.forename: C
authors.initials: C
authors.lastname: Hartnett
authors.forename: M S
authors.initials: MS
authors.lastname: Brown
authors.forename: J
authors.initials: J
authors.lastname: Yu
authors.forename: R J
authors.initials: RJ
authors.lastname: Primus
authors.forename: M
authors.initials: M
authors.lastname: Meyyappan
authors.forename: G
authors.initials: G
authors.lastname: White
authors.forename: V B
authors.initials: VB
authors.lastname: Stirling
authors.forename: J F
authors.initials: JF
authors.lastname: Tallman
authors.forename: T V
authors.initials: TV
authors.lastname: Ramabhadran
authors.forename: D W
authors.initials: DW
authors.lastname: Gallager
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001569:Benzodiazepines / Q000378:metabolism; D001665:Binding Sites; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008024:Ligands; D011725:Pyridines / Q000494:pharmacology; D011963:Receptors, GABA-A / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera; D000077334:Zolpidem; D005680:gamma-Aminobutyric Acid / Q000096:biosynthesis
chemical_list: D008024:Ligands; D011725:Pyridines; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D001569:Benzodiazepines; D005680:gamma-Aminobutyric Acid; D000077334:Zolpidem