Stable expression of cloned rat GABAA receptor subunits in a human kidney cell line.
A predominant form of the GABAA/benzodiazepine receptor-Cl- channel complex is believed to consist of three different 48-55 kDa subunits (alpha, beta, gamma) with unknown stoichiometry. Plasmids containing the rat GABAA receptor cDNAs coding for alpha 1, beta 2, and gamma 2 were co-transfected, along with a plasmid encoding G418 resistance, into human embryonic kidney cells previously transformed with Adenovirus 5 (HEK-293) [J. Gen. Virol., 36 (1977) 59-72]. Four percent of the G418 resistant colonies were found to express mRNA for all three of the GABAA subunits constitutively. A single cell clone derived from one of the alpha 1 beta 2 gamma 2 expressors has demonstrated stable electrophysiological characteristics over 25 passages. The GABA-activated Cl- current in this cell line is blocked by picrotoxin and bicuculline, and is modulated by a variety of agonist and inverse agonist ligands including diazepam, Ro 154513, zolpidem, and beta-CCE. The cell line has been used successfully over a 12-month period as a screen for novel drugs modulating GABA-mediated polarization of neuronal cells.
Hamilton
B J
BJ
CNS Research, Upjohn Company, Kalamazoo, MI 49001.
Lennon
D J
DJ
Im
H K
HK
Im
W B
WB
Seeburg
P H
PH
Carter
D B
DB
D001345:Autoradiography; D015152:Blotting, Northern; D002460:Cell Line; D018118:Chloride Channels; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis; D004594:Electrophysiology; D058787:GABA-A Receptor Antagonists; D006801:Humans; D007473:Ion Channels / Q000187:drug effects; D007668:Kidney / Q000378:metabolism*; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000187:drug effects; D010957:Plasmids; D011963:Receptors, GABA-A / Q000096:biosynthesis*; D014162:Transfection
D018118:Chloride Channels; D058787:GABA-A Receptor Antagonists; D007473:Ion Channels; D008565:Membrane Proteins; D011963:Receptors, GABA-A; D004247:DNA