Five mutations in the GABAA α-6 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 GABA(A) alpha 6-subunit promoter is described in Sardinian alcohol non-preferring rats, selectively bred for their ethanol aversion. These mutations are associated with the R100Q alpha 6 intragenic mutation that was previously characterized in the same animals. The possibility that these mutated nucleotides alter the ethanol-induced upregulation of the a6 gene was investigated by measuring cerebellar alpha 6 mRNA levels after a chronic ethanol liquid diet in sNP rat. Real-time quantitative PCR showed an increased alpha 6 gene expression after ethanol ingestion in normal and mutated rats. However, lower amounts of alpha 6 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 alpha 6 regions comprising the five mutations. These results suggest that one or more of the mutated binding sites that were found in the 5' flanking alpha 6 region may be a consensus sequence for regulatory factors which are responsible for both basal and ethanol-induced alpha 6 gene expression. (c) 2005 Elsevier B.V. All rights reserved.