Development of Stable Cell Lines Expressing Different Subtypes of Gaba_AReceptors

Abstract

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.

Structural Elements of the γ-Aminobutyric Acid Type A Receptor Conferring Subtype Selectivity for Benzodiazepine Site Ligands

“…(Table I). CL218872 is 3-10 times more selective for ␣ 1 -than for ␣ 5 -containing subtypes, and zolpidem is over 300 times more selective (Table I) (17,22,23).…”

“…Cloned cDNAs encoding the ␣ 1 , ␣ 5 , ␤ 2 , and ␥ 2 subunits of rat GABA A receptor were supplied by Prof. P. H. Seeburg (University of Heidelberg) and were subcloned into expression vectors as described previously (17).…”

“…[ 3 H]Flumazenil Equilibrium Binding Assays-Binding assays were performed as described previously (17). Briefly, 50 g of membrane preparation from the transfected cells were incubated with different concentrations of [ Electrophysiology-We used the whole cell configuration of the patch-clamp technique with symmetrical chloride conditions (pipette solution composition was 140 mM CsCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 11 mM EGTA, 10 mM Hepes/Tris-OH, pH 7.2, 4 mM Na 2 ATP; external solution was 147 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 10 mM Hepes/Tris-OH, pH 7.4).…”

Modulation of Recombinant GABA_A Receptors by Neurosteroid Dehydroepiandrosterone Sulfate

“…In this study, stably transfected HEK 293 cells [24] were used to explore the long-term effects of the neurosteroid DHEAS on the recombinant ␣ 1 ␤ 2 ␥ 2S GABA A receptors, the most common type of GABA A receptor found in the brain [25] . However, in order to confirm the specific interactions of DHEAS with ␣ 1 ␤ 2 ␥ 2S GABA A receptors expressed in HEK 293 cells, we first compared the parameters of [ GABA A receptors range between 6 and 7 pmol/mg protein.…”

Chronic exposure of cells expressing recombinant GABAA receptors to benzodiazepine antagonist flumazenil enhances the maximum number of benzodiazepine binding sites

“…Such studies might be important since flumazenil is not only being used acutely to reverse the adverse effects of excessive sedation with benzodiazepines, but it is also being considered for the chronic treatment of hepatic encephalopathy patients (Als-Nielsen et al, 2001;Goulenok et al, 2002;Dursun et al, 2003). Therefore, in this study, stably transfected HEK 293 cells (Besnard et al, 1997) were used as a model to study the effects of prolonged flumazenil exposure on the recombinant a 1 h 2 g 2S GABA A receptors, the most common subunit combination of native GABA A receptors found in the mammalian CNS (McKernan and Whiting, 1996), with the aim to better understand the mechanisms that underlie adaptive changes in GABA A receptors following their prolonged exposure to drugs.…”