Five mutations in the GABAA α6 gene 5′ flanking region are associated with a reduced basal and ethanol-induced α6 upregulation in mutated Sardinian alcohol non-preferring rats

The presence of four nucleotide changes and a three base-pair deletion in the GABAA α6-subunit promoter is described in Sardinian alcohol non-preferring rats, selectively bred for their ethanol aversion. These mutations are associated with the R100Q α6 intragenic mutation that was previously characterized in the same animals. The possibility that these mutated nucleotides alter the ethanol-induced upregulation of the α6 gene was investigated by measuring cerebellar α6 mRNA levels after a chronic ethanol liquid diet in sNP rat. Real-time quantitative PCR showed an increased α6 gene expression after ethanol ingestion in normal and mutated rats. However, lower amounts of α6 mRNA levels were detected both in control and in ethanol-treated sNP rats carrying the five promoter and the intragenic mutations in a homozygous state. Using the electromobility shift assay, specific DNA binding sites were found in cerebellar extracts of the α6 regions comprising the five mutations. These results suggest that one or more of the mutated binding sites that were found in the 5′ flanking α6 region may be a consensus sequence for regulatory factors which are responsible for both basal and ethanol-induced α6 gene expression.