Ethanol exerts profound acute and chronic effects onreceptors for amino acid neurotransmitters in the CNS. In particular, acute exposure to ethanol potentiates the ganna-aminobutyric acid type-A receptor (GABAAR) mediated response, while chronic exposure alters the pharmacology and density of GABAARs in animal models and inpost-mortem brain from alcoholics. It has been proposed that chronic ethanol may cause a switch in the expression of receptor subunit mRNAs, producing an isoform(s) with altered pharmacolgical and physiological properties. However, the mechanism by which receptor function is altered remains unknown yet crucial to our understanding of ethanol tolerance and dependence. During the course of our investigation into the identification and function of promoters for human GABAAR subunit genes, we discovered that ethanol can stimulate (by about 20-fold) alpha1 promoter activity in transfected primary rat neocortical cultures, and that a 63 bp region located in the proximal promoter is crucial for ethanol regulation. The activity of the beta1 promoter is reduced by ethanol, suggesting that one or more cis-regulatory elements are involved in the cell's adaptive response to ethanol. A major objective of this project will be to test our working hypothesis that ethanol interacts with cell surface receptors and exerts genomic regulatory effects through a cellular signal transduction system(s), activating or inhibiting transcription factors which bind to ethanol reposive cis-elements. We have discovered that GABAAR gene diversity arose fromm the duplication and subsequent translocation of an ancestral human alpha-alpha-beta-gamma gene cluster, spawning the clusters on chromosomes 4, 5 and 15. It is our hypothesis that the close evolutionary relationsip of genes within and across clusters predicts coordinate regulation regulation of related genes in different clusters by ethanol. To examine the above hypotheses, ethanol responsive elements in alpha, beta and gamm subunit gene promoters will be identified functionally, using primary neuronal cultures transfected with luciferase reporter constructs containing various deletions and point mutations. We will compare the sequence and fucntion of ehtanol responsive elements from different GABAAR subunit promoters to identify conserved regulatory regions and shared transcription factors. Inhibitors and activators of intracellular messengers will be used to determine the cellular response system that couples receptor modulation to transcriptional regulation. Collectively, these experiments will add to our understanding of the neuron's adaptive response(s) to chronic ethanol exposure, and, in the long-run to the development of diagnostiv and therapeutic agents.
