Stable expression of type I gamma-aminobutyric acidA/benzodiazepine receptors in a transfected cell line.
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.
Wong
G
G
Laboratory of Neuroscience, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Sei
Y
Y
Skolnick
P
P
D000818:Animals; D001386:Azides / Q000378:metabolism; D001569:Benzodiazepines / Q000378:metabolism; D015152:Blotting, Northern; D015139:Blotting, Southern; D002460:Cell Line; D005445:Flunitrazepam / Q000378:metabolism; D006801:Humans; D008297:Male; D010957:Plasmids; D051381:Rats; D017207:Rats, Sprague-Dawley; D011963:Receptors, GABA-A / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
D001386:Azides; D011963:Receptors, GABA-A; D001569:Benzodiazepines; D005445:Flunitrazepam; C042957:Ro 15-4513