MOLECULAR MODELS OF INHALED ANESTHETIC BINDING SITE

我们的目标是使用分子模型来了解麻醉剂分子和它们的结合部位之间的相互作用。为了实现这一点，我们将建立新的假定作用部位的分子模型，计算测试化合物与这些部位的相对结合能，并将结合能与麻醉剂效力关联起来。以下目标将整合这些知识，以确定麻醉剂相互作用的部位，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Harris小组研究的甘氨酸α1和GABA配基门控离子通道跨膜结构域的分子模型。这将为确定假定的麻醉剂结合部位(目标2)提供坚实的基础。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α1亚单位中的Ser267和Ala288)将我们的注意力引导到这些受体的跨膜片段2和3(TM2和TM3)上。TM2和TM3将被建模为α-螺旋，最初参考先前甘氨酸、GABA和尼古丁乙酰胆碱受体中跨膜结构域的模型进行定位。我们将把甘氨酸α1的Overduin核磁共振研究产生的信息纳入我们的模型中。TM2和TM3的螺旋基序假说将通过建模和同源甘氨酸α1受体的定点突变相结合(Harris小组)进行验证。丙氨酸扫描突变、电荷反转突变和双半胱氨酸突变氧化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可能的麻醉剂结合部位，并在分子水平上解释麻醉剂与突变受体的相互作用。Harris小组已经确定TM2和TM3中的单一氨基酸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调节吸入麻醉剂效果的特定位置。我们进一步提出，野生型和突变亚基之间相对结合能的差异可以解释麻醉敏感性的变化。我们将使用在特定目标1中生成的TM2和TM3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