Our goal is to use molecular modeling to understand the interactions between anesthetic molecules and their binding sites. To accomplish this, we will build new molecular models of putative sites of action, calculate relative binding energies of test compounds to these sites, and correlate binding energies with anesthetic potency. The following Aims will integrate this knowledge to identify sites of anesthetic interaction and to reduce atomic properties that distinguish anesthetics from non- immobilizers. 1. We will build and evaluate molecular models of the transmembrane domains of glycine alpha1 and GABA ligand-gated ion channels studied by the Harris group. This will provide a firm basis for identification of putative anesthetic binding sites (Aim 2). Model building will be focused and constrained by experimental data. The sensitivity of anesthetic potency to site-directed mutations (e.g., Ser267 and Ala288 in the glycine alpha1 subunit) directs our attention to transmembrane segments 2 and 3 (TM2 and TM3) of these receptors. TM2 and TM3 will be modeled as alpha-helices, initially oriented by reference to previous models of transmembrane domains in glycine, GABA, and nicotine acetylcholine receptors. We will incorporate information produced by the Overduin NMR studies of glycine alpha1 into our models. The hypothesis of helical motifs for TM2 and TM3 will be tested by a combination of modeling and site directed mutagenesis in homomeric glycine alpha1 receptors (Harris group). A combination of alanine-scanning mutagenesis, charge- reversal mutations, and oxidation of double cysteine mutations will be used to refine the initial model. 2. We will use a knowledge-based approach to identify putative anesthetic binding sites and interpret the interaction of anesthetics with mutated receptors at the molecular level. The Harris group has identified single amino acid mutations in TM2 and TM3 that produce differential effects on our set of test compounds. Our hypothesis is that these site-directed mutations identify specific sites that mediate the effects of inhaled anesthetics. We further propose that differences in relative binding energy between wild type and mutated subunits can explain changes in anesthetic sensitivity. We will test of this hypothesis using the TM2 and TM3 models generated in specific Aim 1 as a starting point
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