Alcoholism is the most common form of substance abuse and has enormous economic burden on the United States and more than 100,000 deaths per year, with countless additional examples of contributory medical problems. Such tremendous costs have motivated intensive research efforts to understand how this drug affects brain function. Yet despite these efforts, there is no consensus as to how ethanol acts at a neuronal level, and no effective medical therapeutic treatment for alcohol abuse disorders is currently available. It is known that ethanol enhances the function of GABAA receptor-mediated signaling in brain and that plastic changes in GABAA receptor (GABARs) subunits (most notably the delta subunit) contribute to the behavioral alterations produced by chronic alcohol use and abuse leading to dependence. Previously evidence has been provided that delta GABARs, which give rise to the extrasynaptic or tonic inhibition, are sensitive to modulation by ethanol at low millimolar concentrations achieved in human social drinking. While these findings provide an explanation for decades of accumulated evidence that inhibitory GABARs are involved in mediating EtOH effects, there has been controversy over the unique of alcohol-sensitivity of these receptors in recombinant expression systems. Preliminary data show (for the first time) that recombinant receptors containing delta subunits are not only uniquely sensitive to EtOH and GABA but also that delta co-expression leads to receptors fractions with a rather dramatic (~1000-fold) increase in sensitivity for the GABA structural analogs THIP/gaboxadol and muscimol. The hypothesis is that in recombinant expression systems there are proteins missing that promote efficient expression surface expression of delta-GABARs. Therefore it is intended to use this property of high THIP sensitivity to screen for delta subunit-binding proteins identifid by a state of the art proteomic/mass spectroscopy approach using immune-affinity purified receptors protein, in order to find potential delta subunit partner proteins that promote cell surface and potentially modulate the function in other interesting ways. The fact that the azido group in the imidazobenzodiazepine EtOH antagonist Ro15-4513 is a photoaffinity will allow the identification of amino acid residues in the EtOH/Ro15-4513 binding site on delta GABA receptors. Based on a detailed structural model hypothesis, the exact amino acid residues that form the alcohol site on delta GABARs will be confirmed and verified using recombinant expression and mutagenesis. This work will help lead to a better understanding of how alcohol and sedative-hypnotic drugs affect brain function. The molecular level identification of ethanol targets is a prerequisite for the development of rational therapies to treat alcohol- related cognitive impairment and alcohol addiction.
This project will study the molecular pharmacology of extrasynaptic brain GABAA receptors, a subset of inhibitory neurotransmitter receptors implicated as key targets in both acute alcohol action and in adaptation to chronic alcohol exposure. We will use unique pharmacological properties of this subset of receptors to determine structure and function of the alcohol site of action on these proteins. Validation and better characterization of this class of alcohol targets might enable the development of new therapies to address alcohol addiction and a variety of other diseases e.g., epilepsy, insomnia, and anxiety.
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