Ribozyme-mediated reduction of the GABA(A) receptor alpha1 subunit.
As an approach to understanding the role of the alpha1 subunit of the GABA(A) receptor, ribozymes were designed to reduce expression of this subunit protein by hydrolysis of alpha1 subunit message and antisense inactivation. The ribozyme cleavage sites were selected through homology comparison of all known murine GABA(A) receptor subunits at the amino acid and nucleotide sequence level. Two ribozymes were designed and synthesized: one against the extracellular domain and the other against the cytoplasmic domain. These ribozymes were cloned in a mammalian expression plasmid, pZeoSV2 (+). Cleavage of both extracellular and cytoplasmic domain transcripts by the respective ribozymes was observed when each ribozyme was tested against in vitro transcribed mRNA. The stable cell line, 122, expressing recombinant human GABA(A) alpha1, beta2 and gamma2S subunits of receptor was stably transfected with the cytoplasmic domain ribozyme (cy) alone and with both the cytoplasmic (cy) and extracellular domain (ex) ribozyme expression plasmids. Northern analysis showed a 55-60% reduction of alpha1 mRNA in clones of cells transfected with either the single ribozyme (Cy) or with both ribozymes (EC). The alpha1 protein level was reduced 75% in a stable Cy clone and more than 90% in a stable EC clone when compared with alpha1 expression in 122 cells and the vector transfected (Zeo) cells. Electrophysiological analysis revealed that the GABA(A) receptor properties were very similar in 122 cells and in stable clones in which the subunit protein expression had been greatly reduced. No significant difference was detected in the potentiation of the receptor response by either bretazenil or zolpidem. These data demonstrate the efficacy of the ribozyme approach in dramatically reducing GABA(A) subunit protein levels in transfected cells and identify those elements that will be important to the application of similar ribozymes to knock-down transmitter receptor subunit proteins under inducible promoters in transgenic mice.
Subramaniam
J R
JR
Department of Biology, Georgetown University, Washington, DC 20057-1229, USA.
Corsi
L
L
Vicini
S
S
Whiting
P J
PJ
Neale
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D001570:Benzodiazepinones / Q000494:pharmacology; D002460:Cell Line; D002474:Cell-Free System; D015536:Down-Regulation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D020320:Oligoribonucleotides, Antisense / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D021122:Protein Subunits; D011725:Pyridines / Q000494:pharmacology; D016337:RNA, Catalytic / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011963:Receptors, GABA-A / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D000077334:Zolpidem
D001570:Benzodiazepinones; D020320:Oligoribonucleotides, Antisense; D021122:Protein Subunits; D011725:Pyridines; D016337:RNA, Catalytic; D012333:RNA, Messenger; D011963:Receptors, GABA-A; D011993:Recombinant Fusion Proteins; D000077334:Zolpidem; C054626:bretazenil