Molecular and electrophysiological characterization of a allelic variant of the rat a6 G A B A A receptor subunit

A 1.45 kb DNA sequence encoding the rat or6 GABA A receptor subunit (nucleotides 33-1483) was cloned from a Sprague-Dawley rat brain cDNA library by PCR amplification. Dideoxy sequencing of two individual clones revealed that the nucleotide sequence differed at only one basepair (T '~° --* G) from that published previously. This difference altered the deduced amino acid sequence, producing a conservative amino acid substitution (His 121 ~ Gln). A Gin residue is present at the same location in the bovine a6 subunit. Restriction endonuclease analysis of the total PCR product demonstrated that this variant of the rat a6 subunit was the only allele found in this particular rat brain library; the original allele was not present. These results were further verified by RNAse protection assays performed with RNA isolated from individual rat cerebella, a6,/31, and y2S subunits were transiently expressed in L929 cells for electrophysiological analysis. Whole-cell recordings obtained from the cells demonstrated that GABA A receptor channels with the expected GABA and benzodiazepine pharmacology were produced. Excised outside out single channel recordings from the same cells revealed that GABA elicited brief duration openings to a 33 pS main conductance level and to at least one smaller (approximately 21 pS) subconductance level. Thus this allelic variant of rat a6 subunit could assemble with other subunits to form a functional GABA A receptor channel with similar properties to the original allelic form.