MOLECULAR MODELS OF INHALED ANESTHETIC BINDING SITE

我们的目标是使用分子模拟来了解麻醉剂分子和它们的结合位点之间的相互作用。为了实现这一点，我们将建立新的分子模型的推定作用位点，计算相对结合能的测试化合物对这些网站，并与麻醉效力的结合能。以下目标将整合这些知识，以识别麻醉剂相互作用的位点，并减少区分麻醉剂和非固定剂的原子性质。1.我们将建立和评估跨膜结构域的甘氨酸α 1和GABA配体门控离子通道的哈里斯组研究的分子模型。这将为确定假定的麻醉剂结合位点提供坚实的基础（目的2）。模型的建立将受到实验数据的关注和约束。麻醉效力对定点突变的敏感性（例如，甘氨酸α 1亚基中的Ser 267和Ala 288）将我们的注意力引向这些受体的跨膜片段2和3（TM 2和TM 3）。TM 2和TM 3将被建模为α-螺旋，最初通过参考甘氨酸、GABA和尼古丁乙酰胆碱受体中的跨膜结构域的先前模型来定向。我们将把甘氨酸α 1的Overduin NMR研究产生的信息纳入我们的模型。TM 2和TM 3的螺旋基序的假设将通过在同型甘氨酸α 1受体（Harris组）中建模和定点诱变的组合来测试。丙氨酸扫描诱变、电荷逆转突变和双半胱氨酸氧化突变的组合将用于完善初始模型。2.我们将使用基于知识的方法来确定假定的麻醉剂结合位点，并在分子水平上解释麻醉剂与突变受体的相互作用。哈里斯小组已经确定了TM 2和TM 3中的单个氨基酸突变，这些突变对我们的一组测试化合物产生不同的影响。我们的假设是，这些定点突变确定了介导吸入麻醉剂作用的特定位点。我们进一步提出，野生型和突变亚基之间的相对结合能的差异可以解释麻醉剂敏感性的变化。我们将使用特定目标1中生成的TM 2和TM 3模型作为起点来检验这一假设

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