A combination of human alpha 1 and beta 1 subunits is required for formation of detectable GABA-activated chloride channels in Sf9 cells.
The baculovirus expression system was used to produce alpha 1 and beta 1 subunits of the human GABAA receptor in Sf9 cells. In cells infected with both alpha 1 and beta 1 recombinant viruses, GABA elicited an outwardly rectifying chloride current that was blocked by bicuculline and potentiated by pentobarbitone. GABA did not produce detectable currents in cells infected with either alpha 1 or beta 1 recombinant viruses alone. In these cells, and in control (non-infected) Sf9 cells, pentobarbitone depressed the leakage current (Ki = 55 microM). Fluorescently labelled monoclonal antibodies to the alpha 1 subunit showed greater amounts of the alpha 1 subunit in cells infected with only the alpha 1 recombinant virus than in cells co-infected with the alpha 1 and beta 1 recombinant viruses. Fluorescence of the plasma membrane was seen in cells co-infected with the alpha 1 and beta 1 recombinant viruses, but was absent in cells infected with only the alpha 1 recombinant virus. It was concluded that the alpha 1 subunit normally interacts with the beta 1 subunit to be transported to the plasma membrane in Sf9 cells.
Birnir
B
B
John Curtin School of Medical Research, Australian National University, Canberra ACT.
Tierney
M L
ML
Howitt
S M
SM
Cox
G B
GB
Gage
P W
PW
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D018118:Chloride Channels; D004553:Electric Conductivity / Q000187:drug effects; D005455:Fluorescent Antibody Technique; D006801:Humans; D007313:Insecta; D007473:Ion Channels / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D046911:Macromolecular Substances; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D008856:Microscopy, Fluorescence; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology*
D018118:Chloride Channels; D007473:Ion Channels; D046911:Macromolecular Substances; D008565:Membrane Proteins; D011994:Recombinant Proteins; D005680:gamma-Aminobutyric Acid; D001616:beta-Galactosidase