SMALL N-TERMINAL DELETION BY SPLICING IN CEREBELLAR ALPHA-6 SUBUNIT ABOLISHES GABA(A) RECEPTOR FUNCTION

Sequence variation was found in cDNA coding for the extracellular domain of the rat gamma-aminobutyric acid type A (GABA(A)) 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 GABA, receptor beta 2 and gamma 2 subunits in human embryonic kidney 293 cells, were inactive at binding [H-3]muscimol and [H-3]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.