DEVELOPMENT OF STABLE CELL-LINES EXPRESSING DIFFERENT SUBTYPES OF GABA(A) RECEPTORS

The experiments reported here were motivated by our interest to expres s in stably-transfected cells large amounts of recombinant rat GABA(A) receptors. For this, we developed an original two step selection stra tegy, in which the first step consisted of transfecting HEK 293 cells with rat GABA(A) receptor alpha and beta subunits. G 418 resistant col onies isolated at this step were screened for [H-3] muscimol binding t o 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 mutan t DHFR gene. After a second round of selection, this time in presence of methotrexate, those colonies that coexpressed ternary alpha beta ga mma GABA(A) receptor combinations were distinguished using [H-3] fluma zenil 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) gamm a(2S) and alpha(5) beta(3) gamma(2S), that expressed relatively high l evels of these proteins. These 3 cell lines exhibited pharmacological and functional properties similar to cells transiently-transfected wit h equivalent subunit combinations. These cell lines therefore provide attractive models with which to evaluate the intrinsic activity and po tency of compounds at recombinant GABA(A) receptor subtypes.