Alpha 6 and gamma 2 subunit antisense oligodeoxynucleotides alter gamma-aminobutyric acid receptor pharmacology in cerebellar granule neurons.
To characterize the role of the alpha 6 subunit in gamma-aminobutyric acid (GABA) receptors in cerebellar granule cells, primary cerebellar cultures were treated with antisense oligodeoxynucleotides (ODNs) complementary to and overlapping the initial codon of the alpha 6 subunit cDNA. The specific reduction in the expression of the alpha 6 receptor subunit protein after a 48-hr antisense ODN treatment was assessed with the use of immunoblot assays. Sister cultures were treated in parallel with mismatched (scrambled) ODNs. Inhibition of GABA-gated currents by furosemide, a selective inhibitor of GABAA receptors containing alpha 6 subunits, was attenuated after the alpha 6 antisense treatment. Furosemide was tested in parallel in transfected cells expressing various combinations of the alpha 1 and alpha 6 subunits, which showed that the relative abundance of these subunit mRNAs determines the extent of furosemide-induced inhibition of GABA-gated currents. Compared with control or mismatched ODN-treated cell cultures, treatment of granule neurons with alpha 6 antisense ODNs caused a decrease in GABA-induced maximal current density and increased the half-maximal concentration derived from GABA dose-response curves. Furthermore, the depletion of alpha 6 subunits from cerebellar granule cells enhanced flunitrazepam-induced potentiation of GABA-activated currents. In contrast, gamma 2 antisense ODN treatments of cell cultures increased the receptor sensitivity to GABA and potently decreased the response to flunitrazepam. Our results show that alpha 6 and gamma 2 subunit expression can be blocked with the use of synthetic ODNs and that these subunits are crucial determinants of the pharmacological properties of native GABAA receptors in cerebellar granule cells.
Zhu
W J
WJ
Department of Psychiatry, Medical College of Pennsylvania, Pittsburgh, USA.
Wang
J F
JF
Vicini
S
S
Grayson
D R
DR
D000494:Allosteric Regulation; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D002531:Cerebellum / Q000502:physiology*; D003062:Codon; D018076:DNA, Complementary; D005665:Furosemide / Q000494:pharmacology; D006801:Humans; D007668:Kidney; D007700:Kinetics; D046911:Macromolecular Substances; D008564:Membrane Potentials / Q000187:drug effects; D008969:Molecular Sequence Data; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley; D018079:Receptors, GABA / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D014162:Transfection; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology*
D003062:Codon; D018076:DNA, Complementary; D046911:Macromolecular Substances; D016376:Oligonucleotides, Antisense; D018079:Receptors, GABA; D005680:gamma-Aminobutyric Acid; D005665:Furosemide