Cloned GABA receptors are maintained in a stable cell line: allosteric and channel properties.
The cloned cDNAs encoding the alpha 1 and beta 1 subunits of the bovine brain GABA(A) receptor have been co-transfected, using a dexamethasone-inducible promoter, into cultured hamster ovary cells, with selection to form a stable cell line. The use, alternatively, of a much stronger constitutive promoter led to cell death consequent upon high receptor density. After induction, the cells contained the alpha 1 and beta 1 mRNAs. The expressed receptors showed the high-affinity binding of [3H]muscimol and of the GABA(A) receptor channel blocker, t-butylphosphorothionate (TBPS), and the characteristic enhancement of the former by a pregnanolone. Their GABA-activated current was potentiated by the barbiturate, pentobarbitone, was reversibly blocked by bicuculline and picrotoxin, but was not enhanced by benzodiazepines. In mouse spinal cord neurons GABA activates channel openings to at least four conductance states (45, 30, 19 and 12 pS) with the 30 pS state being the most frequently observed (main) state. However, the main state of the alpha 1/beta 1 GABA(A) receptor was the 19 pS state. The enhancement of GABA(A) receptor current by barbiturates wa due to prolongation of mean channel lifetime, whereas the reduction of GABA(A) receptor current by picrotoxin was due to reduction of channel opening frequency and mean channel lifetime. Stable cell lines containing subunit combinations of this receptor should provide a powerful tool for the elucidation of its channel features and control mechanisms.
Moss
S J
SJ
MRC Molecular Neurobiology Unit, MRC Centre, Cambridge, U.K.
Smart
T G
TG
Porter
N M
NM
Nayeem
N
N
Devine
J
J
Stephenson
F A
FA
Macdonald
R L
RL
Barnard
E A
EA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001463:Barbiturates / Q000378:metabolism; D001640:Bicuculline / Q000378:metabolism; D001643:Bridged Bicyclo Compounds / Q000378:metabolism; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002417:Cattle; D002460:Cell Line / Q000502:physiology; D002712:Chlorides / Q000502:physiology; D002999:Clone Cells / Q000502:physiology; D003292:Convulsants / Q000378:metabolism; D004247:DNA / Q000235:genetics; D008564:Membrane Potentials / Q000502:physiology; D009118:Muscimol / Q000378:metabolism; D009474:Neurons / Q000648:ultrastructure; D010957:Plasmids; D011963:Receptors, GABA-A / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D013116:Spinal Cord / Q000166:cytology; D014162:Transfection; D005680:gamma-Aminobutyric Acid / Q000502:physiology
D000911:Antibodies, Monoclonal; D001463:Barbiturates; D001643:Bridged Bicyclo Compounds; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002712:Chlorides; D003292:Convulsants; D011963:Receptors, GABA-A; D009118:Muscimol; D005680:gamma-Aminobutyric Acid; C037476:tert-butylbicyclophosphorothionate; D004247:DNA; D001640:Bicuculline