Ethanol exerts its acute effects on the mammalian central nervous system (CNS), at least in part, by altering the function of gamma-aminobutyric acid GABA/a)-activated chloride channels (hence forward called GABA channels). Molecular cloning studies have so far identified 12 distinct GABA channel subunit isoforms and these isoforms are differentially distributed in the CNS. This suggests different regions of the brain assemble GABA channels from different combinations of subunits. Prior to identification of the various isoforms, GABA channel heterogeneity was suggested by the observation of functional subtypes (electrophysiology), receptor subtypes (ligand binding), and apparent size subtypes (electrophoresis). This raises the possibility that ethanol, by having differential effects on the various GABA channels, might exert differential effects on different brain regions. This project will combine molecular biological and electrophysiological techniques and test this 'differential- regulation' hypothesis by examining the actions of ethanol on GABA channels of different subunit combinations. cDNAs encoding the GABA channel subunits will be transcribed in-vitro and the cRNA will be injected, in various combinations, into frog oocytes. Two-electrode voltage clamp techniques will then be used to assess ethanol's modulation of currents through the various GABA channels. Another fundamental question regarding the actions of ethanol on GABA channels is the mechanism by which ethanol modulates GABA channels. Ethanol could modulate either the ion-permeation properties or the opening and closing kinetics (gating) of the GABA channel. Modulation of gating, the more likely possibility, could result from a variety of mechanisms, one example being a change in the stability of the open-pore conformation of the channel. To test these and other putative modulatory mechanisms, the patch-clamp technique will be used to record single GABA-activated ion channels constructed from subunit combinations found to be ethanol sensitive. A detailed analysis of single-channel data recorded in the absence and presence of ethanol will suggest molecular mechanism(s) for ethanol's modulation of GABA channels and will enable us to compare how ethanol modulates GABA channels composed of different subunits. The long term objectives of this project are to determine the mechanisms by which ethanol modulates GABA-activated ion channels in the CNS. This information will increase our understanding of fundamental aspects of ethanol's actions on synaptic inhibition in the CNS, and facilitate the designing and testing of drugs to antagonize the intoxicating effects of ethanol and treat various ethanol-related diseases, such as alcohol withdrawal syndrome. Furthermore, comparison of the interactions of ethanol with GABA channels of different subunit composition is a first step towards identifying ethanol's specific sites of action on the GABA channel.
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