GABAA RECEPTOR BETA-SUBUNIT AND GENERAL ANESTHETICS

A型γ氨基丁酸(GABAA)受体是一种 全麻药的重要药理作用靶点。最新进展 提示几种全身麻醉药在GABAA的高度特异性作用 受体；然而，这种机制的细节尚不清楚。另外 组成GABAA的亚基有许多不同的形式 受体。GABAA受体亚基组成与GABAA的相互作用 全身麻醉药理学仍然相对未被探索，而 这种相互作用对神经元功能的意义尚不清楚。这 该项目测试了GABAA受体β亚基亚型的假设 决定依托咪酯对GABA诱导的神经元电流的调制。 这项研究由一系列深思熟虑的步骤组成，从生物物理开始 静脉全麻药在GABAA中的作用特征 受体，然后检查b亚基的作用 基因打靶技术在全身麻醉药理学研究中的应用 创造具有可逆性外部诱导改变的神经元 对依托咪酯敏感。具体来说，最近发现的关键角色 B亚基和位于第270位的特定氨基酸 β亚基将通过在HEK293细胞中表达不同的 A1bxg2异构体中的β亚基异构体和点突变。 全细胞膜片钳和快速GABA灌流将用于 药物作用的动力学模型。接下来，这个机械模型将被用作 研究β亚基基因靶向对一般情况的影响的工具 维甲酸诱导的P19神经元的麻醉药理学。P19系统 为研究基因靶向的影响提供了一个前所未有的机会 没有创造一整只动物所涉及的复杂性。传统型 B1转基因、b1基因敲除和条件性基因靶向神经元 检查过了。在研究项目的下一阶段，最好的基因靶向 在这些研究中发现的策略将被用于创造一种基因靶向的小鼠 具有可逆的外部诱导的对一般情况的敏感性改变 麻醉剂。 详细了解现有的全身麻醉药的工作原理是至关重要的 对于开发改进的麻醉药来说没有非常重要的一面 现有药剂的影响。拟议的实验将增加数量和 全麻药分子药理学的机理信息及其进展 这一领域的调查更接近于真正了解这些 临床基本药物有效。

The type A gamma amino butyric acid (GABAA) receptor is an important pharmacological target for general anesthetic drugs. Recent progress suggests highly specific effects of several general anesthetics at the GABAA receptor; the details of this mechanism, however, are unknown. Additionally there are many different forms of the subunits which make up the GABAA receptor. The interaction between GABAA receptor subunit composition and general anesthetic pharmacology remains relatively unexplored, and the significance of this interaction to the function of neurons is unknown. This project tests the hypothesis that the GABAA receptor beta-subunit isoform dictates etomidate modulation of GABA-induced current in neurons. The study consists of a series of deliberate steps beginning with a biophysical characterization of the effects of intravenous general anesthetics at GABAA receptors, followed by an examination of the effects of b-subunit gene-targeting on general anesthetic pharmacology and concluding with the creating of neurons with a reversible externally-inducible alteration in sensitivity to etomidate. Specifically, the recently discovered critical role of the beta-subunit and the specific amino acid at location 270 on the beta-subunit will be explored through expression, in HEK293 cells, of different beta-subunit isoforms and point-mutants in an a1bxg2 heteromeric combination. Whole cell patch clamp and rapid GABA perfusion will be used to arrive at a kinetic model of drug action. Next this mechanistic model will be used as a tool to investigate the effects of beta-subunit gene-targeting on general anesthetic pharmacology in retinoic-acid-induced P19 neurons. The P19 system offers an unprecedented opportunity to investigate the effect of gene-targeting without the complexities involved in creating a whole animal. Conventional b1-transgenic, b1-knockout, and conditional gene-targeted neurons will be examined. In the next phase of the research project, the best gene-targeting strategy found in these studies will be used to create a gene-targeted mouse with a reversible externally-inducible alteration in sensitivity to general anesthetics. Detailed understanding of how existing general anesthetics work is essential for the development of improved anesthetics without the very significant side effects of existing agents. The experiments proposed will add quantitative and mechanistic information to general anesthetic molecular pharmacology and move this field of investigation closer towards truly understanding how these clinically essential drugs work.