Development of stable cell lines expressing different subtypes of GABA(A) receptors

The experiments reported here were motivated by our interest to express in stably-transfected cells large amounts of recombinant rat GABA(A) receptors. For this, we developed an original two step selection strategy, in which the first step consisted of transfecting HEK 293 cells with rat GABA(A) receptor alpha and beta subunits. G 418 resistant colonies isolated at this step were screened for [H-3] muscimol binding to select for those that coexpressed alpha- and beta-subunits. The best a and beta subunit expressing colony was then supertransfected with a plasmid coding for the gamma rat GABA(A) 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 GABA(A) receptor combinations were distinguished using [H-3] flumazenil as a probe. This strategy was applied to the isolation of 3 GABA(A) 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 GABA(A) receptor subtypes.