PT  - JOURNAL ARTICLE
AU  - G Wong
AU  - Y Sei
AU  - P Skolnick
TI  - Stable expression of type I gamma-aminobutyric acidA/benzodiazepine receptors in a transfected cell line.
DP  - 1992 Dec 01
TA  - Molecular Pharmacology
PG  - 996--1003
VI  - 42
IP  - 6
4099  - http://molpharm.aspetjournals.org/content/42/6/996.short
4100  - http://molpharm.aspetjournals.org/content/42/6/996.full
SO  - Mol Pharmacol1992 Dec 01; 42
AB  - Expression plasmids were constructed with cDNAs encoding the rat gamma-aminobutyric acid-A (GABAA) receptor alpha 1, beta 2, and gamma 2 subunits and were cotransfected into cultured human embryonic kidney 293 cells. A single cell line (WS-1) was established after G-418 treatment and clonal selection. This cell line contained saturable, high affinity binding sites for the benzodiazepines [3H] Ro 15-4513 and [3H]flunitrazepam that were modulated by GABA. Competition experiments with benzodiazepine receptor ligands suggest a profile characteristic of native "type I" benzodiazepine receptors, because strong correlations were observed between the Ki values of these ligands in WS-1 cells and in both cerebellar homogenates (r = 0.97, p < 0.0001) and 293 cells transiently transfected with the corresponding cDNAs (r = 0.96, p < 0.001). Fluorescence intensity in WS-1 cells loaded with the Cl(-)-specific probe 6-methoxy-N-(3-sulfopropyl)-quinolinium was reliably increased by GABA. This effect was blocked by bicuculline and augmented by midazolam, consistent with the presence of GABA-gated, benzodiazepine receptor-modulated, Cl- channels. Northern blot analysis revealed the presence of mRNAs encoding alpha 1 and gamma 2 receptor subunits. Southern blot analysis confirmed genomic integration of transfected alpha 1 and gamma 2 cDNAs. The beta 2 subunit was not detected in either Northern or Southern blot analysis, indicating that a functional type I GABAA/benzodiazepine receptor complex can be constituted without a beta subunit.