Small N-terminal deletion by splicing in cerebellar alpha 6 subunit abolishes GABAA receptor function.
Sequence variation was found in cDNA coding for the extracellular domain of the rat gamma-aminobutyric acid type A (GABAA) receptor alpha 6 subunit. About 20% of polymerase chain reaction (PCR)-amplified alpha 6 cDNA prepared from rat cerebellar mRNA lacked nucleotides 226-255 as estimated by counting single-stranded phage plaques hybridized specifically to the short (alpha 6S) and long (wild-type) forms of the alpha 6 mRNA. Genomic PCR revealed an intron located upstream of the 30-nucleotide sequence. Both splice forms were detected in the cerebellum by in situ hybridization. Recombinant receptors, resulting from coexpression of the alpha 6S subunit with the GABAA receptor beta 2 and gamma 2 subunits in human embryonic kidney 293 cells, were inactive at binding [3H]muscimol and [3H]Ro 15-4513. In agreement, injection of complementary RNAs encoding the same subunits into Xenopus oocytes produced only weak GABA-induced currents, indistinguishable from those produced by beta 2 gamma 2 receptors. Therefore, the 10 amino acids encoded by the 30-nucleotide fragment may be essential for the correct assembly or folding of the alpha 6 subunit-containing receptors.
Korpi
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ER
Laboratory of Molecular Neuroendocrinology, University of Heidelberg, Germany.
Kuner
T
T
Kristo
P
P
Köhler
M
M
Herb
A
A
Lüddens
H
H
Seeburg
P H
PH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002531:Cerebellum / Q000737:chemistry*; D005260:Female; D017353:Gene Deletion; D018014:Gene Transfer Techniques; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D007668:Kidney; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012326:RNA Splicing; D051381:Rats; D018079:Receptors, GABA / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D014981:Xenopus; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D018079:Receptors, GABA; D011994:Recombinant Proteins; D005680:gamma-Aminobutyric Acid