Expression of a bovine GABAA receptor alpha1-subunit cDNA in murine erythroleukaemia cells.
A plasmid vector has been constructed by insertion of the cDNA encoding the alpha1 subunit of the bovine GABAA receptor into the LCR/MEL expression vector pNV1 downstream of the human globin locus control region between the promoter and the second intron of the beta-globin gene to produce pNVGABAalpha. This plasmid was transfected into murine erythroleukemia (MEL) cells using electroporation to obtain recombinant cells. Parental and recombinant cells were tested by both RNA dot blot and electrophysiological analysis for the presence of bovine GABAA receptor alpha1 subunit mRNA. Parental MEL cells did not express GABA-gated chloride channels but recombinant cells were sensitive to pressure-applied GABA. The GABA responses reversed at the equilibrium potential predicted for chloride ions. These results show that the alpha1 subunit of the bovine GABAA receptor inserts in the plasma membrane of the MEL cells and forms homo-oligomeric chloride channels that are gated by GABA. Our studies suggest, therefore, that the LCR/MEL system can be used for the expression of neurotransmitter receptor genes.
School of Biological and Molecular Sciences, Oxford Brookes University, UK.
M
M
Amar
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L
Smith
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M
Needham
D J
DJ
Beadle
L A
LA
King
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I
Bermudez
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels / Q000187:drug effects / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D004915:Leukemia, Erythroblastic, Acute / Q000235:genetics / Q000378:metabolism; D051379:Mice; D011487:Protein Conformation; D011963:Receptors, GABA-A / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D018118:Chloride Channels; D018076:DNA, Complementary; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D005680:gamma-Aminobutyric Acid