Development of stable cell lines expressing different subtypes of GABAA receptors.
The experiments reported here were motivated by our interest to express in stably-transfected cells large amounts of recombinant rat GABAA receptors. For this, we developed an original two step selection strategy, in which the first step consisted of transfecting HEK 293 cells with rat GABAA receptor alpha and beta subunits. G 418 resistant colonies isolated at this step were screened for [3H] muscimol binding to select for those that coexpressed alpha- and beta-subunits. The best alpha and beta subunit expressing colony was then supertransfected with a plasmid coding for the gamma rat GABAA receptor subunit and a mutant DHFR gene. After a second round of selection, this time in presence of methotrexate, those colonies that coexpressed ternary alpha beta gamma GABAA receptor combinations were distinguished using [3H] flumazenil as a probe. This strategy was applied to the isolation of 3 GABAA receptor clones, alpha 1 beta 2 gamma 2s, alpha 3 beta 2 gamma 2s and alpha 5 beta 3 gamma 2s, that expressed relatively high levels of these proteins. These 3 cell lines exhibited pharmacological and functional properties similar to cells transiently-transfected with equivalent subunit combinations. These cell lines therefore provide attractive models with which to evaluate the intrinsic activity and potency of compounds at recombinant GABAA receptor subtypes.
Besnard
F
F
Synthélabo Recherche, Rueil Malmaison, France.
Even
Y
Y
Itier
V
V
Granger
P
P
Partiséti
M
M
Avenet
P
P
Depoortere
H
H
Graham
D
D
D000818:Animals; D001569:Benzodiazepines / Q000378:metabolism; D001667:Binding, Competitive; D002460:Cell Line; D018118:Chloride Channels / Q000378:metabolism; D004594:Electrophysiology; D005442:Flumazenil / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D011487:Protein Conformation; D051381:Rats; D011963:Receptors, GABA-A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014162:Transfection
D018118:Chloride Channels; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D001569:Benzodiazepines; D005442:Flumazenil; D013762:Tetrahydrofolate Dehydrogenase