Activation of glycine receptors (GlyRs) in brain stem, spinal cord and brain provides a main inhibitory control for neuronal excitability. Modification of these receptors by toxins and drugs such as ethanol leads to a wide range of effects varying from sedation and muscle relaxation to convulsions, respiratory arrest and death. However, the mechanism by which ethanol affects these receptors is largely unknown. Recent studies have indicated that GlyR function is modified by changes in GTP-binding protein activity. Specifically, using a series of GlyR mutants, the presence of basic amino acid motifs that are responsible for G beta gamma binding and receptor modulation were discovered. More importantly, preliminary results show that basic amino acids in the main intracellular loop of the GlyR determine the sensitivity of the receptor to clinically relevant concentrations of ethanol (10-100 mM). These amino acids also regulate the interaction of the GlyR with G beta gamma. In agreement with these findings, it was found that GlyR sensitivity to ethanol was significantly modified by changing the stoichiometry of the heterotrimeric complex. Therefore, it was hypothesized that important determinants for ethanol sensitivity are within the intracellular loop and that the sensitivity of GlyR to ethanol depends on the state of G protein activation. Remarkably, it was shown that these sites did not participate in the modulation of GlyRs by propofol, isoflurane and long chain alcohols indicating a novel and selective new site for ethanol. Additionally, mutations in these sites did not affect the physiological properties of the GlyR indicating that the ethanol resistant phenotype was not related to changes on channel gating. Therefore, the primary objective of this study is to characterize the intracellular sites responsible for G protein activation and GlyR sensitivity to ethanol by using receptor mutagenesis, kinetic analysis of GTP binding to G proteins, heterologous expression of G protein subunits, patch-clamp and Western blot techniques. In addition, key experiments will be performed using WT and mutant GlyRs expressed in a neuronal background (i.e. DRG neurons). The information obtained from this study will help to understand the mechanism by which ethanol affects glycine receptors, which are important in functions such as convulsions, sensory integration, muscle tone and respiration. Therefore, these results may contribute to the development of new treatments for alcoholism. PUBLIC HEALTH RELEVANCE Excessive consumption of ethanol is a main health-related problem in the US and world wide. However, the main determinants for the changes in the brain functions produced by ethanol are still largely unknown. Therefore, it is important to identify the mechanism and sites of ethanol action in order to understand how ethanol affects the central nervous system and to propose useful therapies to alleviate neurological health problems associated with its excessive consumption.