Alcohol abuse and alcoholism are major health problems. It is likely that a solution to these problems will require an understanding of the effects of alcohol on neuronal ion channel proteins. Specific binding sites for alcohols have been described in the transmembrane (TM) domain of the superfamily of ligand-gated ion channels (LGIC's) that includes glycine (GlyRa1), GABA, nicotinic acetylcholine, and 5-HT3 receptors. Our global hypothesis is that alcohols bind within cavities that are bounded by TM segments of these receptors and preferentially stabilize specific channel substates. Our goal is to define the properties of those sites that mediate binding and efficacy of alcohol and alcohol antagonists in GlyRa1. These binding sites may provide a common motif for binding of alcohols within other classes of ion channels and other important proteins. We will build computational models of binding sites in GlyRa1 and design specific site-directed mutations to test these hypothesis. These mutations will be constructed and tested by our collaborator, Dr. R. A. Harris.
Aim 1. We will test the hypothesis that amino acid residues from all four transmembrane helices of GlyRa1 contribute to a binding site for alcohols. We will develop computational models to delineate the dimensions of these sites. We will use these models to predict where covalent binding of alkyl methanethiosulfonate (MTS) reagents would mimic alcohol binding. The predictions will be tested in the Harris laboratory by expressing GlyRa1 containing site-directed cysteine substitutions in oocytes. They will apply MTS reagents to the oocytes and measure the effects on glycine-induced currents.
Aim 2. We will test the hypothesis that double site directed cysteine mutations can clarify the refined tertiary structure of the GlyRa1 and distinguish our model from one based on the torpedo nAChR. While the overall structure of our GlyRa1 model and the nAChR model of Unwin are in global agreement, there are important differences in the orientation of transmembrane helices and residues bounding a possible alcohol- binding site. The Harris laboratory will test these predictions by crosslinking site-directed di-cysteines. In summary, knowledge of alcohol binding sites in GlyRa1 will increase our understanding of alcohol action in LGIC's. The results may reveal general motifs for action in other systems affected by alcohol and aid in the design of alcohol antagonists.
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