Comparison of interactions of [3H]muscimol, t-butylbicyclophosphoro[35S]thionate, and [3H]flunitrazepam with cloned gamma-aminobutyric acidA receptors of the alpha 1 beta 2 and alpha 1 beta 2 gamma 2 subtypes.
The alpha 1 beta 2 and alpha 1 beta 2 gamma 2 subtypes, common subtypes of gamma-aminobutyric acid (GABA)A receptors in the brain, are known to share many ligands, but only the latter interacts with benzodiazepines. In this study, we attempted to examine whether the presence of the gamma 2 subunit in the cloned receptors alters the binding properties of GABA and t-butylbicyclophosphorothionate (TBPS) (a highly sensitive probe for conformational changes in the chloride ionophore of GABAA receptors) and their interactions. Using a high-level expression system of SF-9 cells infected with baculovirus, we produced a group of cloned GABAA receptors with variations in the ratio (0 to 3) of the virion carrying the cDNA for the gamma 2 subunit to those carrying the cDNAs for the alpha 1 and beta 2 subunits. The number of benzodiazepine binding sites increased as the level of the gamma 2 virion was raised and reached that of GABA high affinity sites at a gamma 2 to alpha 1 beta 2 ratio of 0.5 or more. It appears that the conversion of the alpha 1 beta 2 to the alpha 1 beta 2 gamma 2 subtype is favorable and complete in the presence of a sufficient level of the gamma 2 subunit, assuming the number of the GABA sites to be equal to the total number of the cloned GABAA receptors. In all preparations, the dissociation constants for flunitrazepam, muscimol, and TBPS were fairly constant, and the maximal number of binding sites for TBPS appeared to be equal to that for muscimol, with no dependence on the gamma 2 virion levels. The effect of GABA on TBPS binding, however, were markedly altered by the gamma 2 subunit. With the alpha 1 beta 2 subtype GABA at concentrations occupying its high affinity sites markedly stimulated but at higher concentrations (micromolar ranges) inhibited TBPS binding, whereas with the alpha 1 beta 2 gamma 2 subtype GABA inhibited TBPS binding without the early stimulatory phase. We also confirmed the selective interaction of Zn2+ (50 microM) with the alpha 1 beta 2 subtype, as probed with TBPS binding, and observed a progressive disappearance of Zn2+ sensitivity as the gamma 2 virion level increased.(ABSTRACT TRUNCATED AT 400 WORDS)
Upjohn Company, CNS Diseases Research, Kalamazoo, Michigan 49001.
J F
JF
Pregenzer
W B
WB
Im
D B
DB
Carter
D R
DR
Thomsen
D000818:Animals; D001643:Bridged Bicyclo Compounds / Q000378:metabolism*; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002460:Cell Line; D003001:Cloning, Molecular; D005445:Flunitrazepam / Q000378:metabolism*; D009036:Moths; D009118:Muscimol / Q000378:metabolism*; D011869:Radioligand Assay; D011963:Receptors, GABA-A / Q000145:classification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D001643:Bridged Bicyclo Compounds; D019086:Bridged Bicyclo Compounds, Heterocyclic; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D009118:Muscimol; D005445:Flunitrazepam; C037476:tert-butylbicyclophosphorothionate; D015032:Zinc