Ethanol sensitivity--native /recombinant NMDA receptors

乙醇敏感性——天然/重组 NMDA 受体

• 批准号: 6930609
• 负责人: JOHN J. WOODWARD
• 金额: $ 15.69万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-04-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6930609
• 关键词: NMDA receptors; Xenopus oocyte; calcium flux; cell line; electrophysiology; ethanol; genetically modified animals; human genetic material tag; inhibitor /antagonist; laboratory rat; neurons; neuropharmacology; neuropsychology; neurotransmitter transport; postdoctoral investigator; protein structure; receptor expression; receptor sensitivity; recombinant proteins; site directed mutagenesis; tissue /cell culture; transfection; voltage /patch clamp

DESCRIPTION (provided by applicant): This application is a competitive renewal of a currently funded K02 award and is focused on advancing the PI's ability to investigate the actions of ethanol on brain ion channels. The PI has conducted research into the effects of alcohol of the NMDA receptor and is currently funded by NIAAA through an R01 to continue this work. The PI has utilized the currently funded K02 award period to obtain training in two-electrode voltage-clamp, whole-cell patch-clamp eletrophysiology and molecular biology. He has used these techniques to make important discoveries with regard to the molecular sites of alcohol action of the NMDA receptor. Results from experiments utilizing transfection of oocytes and HEK293 cells with wild-type and mutant NMDA receptors has led to a better understanding of how subunit assembly, intracellular signaling processes and transmembrane domains modulate the sensitivity of this receptor to alcohol. In this application, the PI will extend the relevance of these findings by learning techniques used to express mutant NMDA receptor subunits with altered ethanol sensitivity in brain neurons and to assess specific areas of molecular biology, electrophysiology, and animal behavior. Specifically, the PI will: 1) learn techniquesneeded to generate and use viral-based gene vectors to deliver NMDA receptor subunits with altered ethanol sensitivity into neurons grown in culture and in vivo; 2) learn methods used to generate genetically modified animals that express mutant NMDA receptor subunits with altered ethanol sensitivity; 3) learn methods to prepare and record ion channel currents from brain slices isolated from animals expressing mutant NMDA receptors with altered ethanol sensitivity and; 4) learn techniques needed to assess the neurobehavioral actions of alcohol in animals expressing mutant NMDA receptors with altered ethanol sensitivity. This training will be obtained during a series of sessions supervised by a variety of consultants with demonstrated expertise in the techniques. The successful completion of this training plan will substantially enhance the PI's research expertise and his ability to fulfill the goals of his research.

描述（由申请人提供）： 该申请是目前资助的K 02奖的竞争性更新，并专注于推进PI研究乙醇对脑离子通道作用的能力。 PI已经对酒精对NMDA受体的影响进行了研究，目前由NIAAA通过R 01资助继续这项工作。 PI利用目前资助的K 02奖励期获得了双电极电压钳、全细胞膜片钳电生理学和分子生物学方面的培训。 他利用这些技术在NMDA受体的酒精作用的分子位点方面做出了重要发现。 利用野生型和突变型NMDA受体转染卵母细胞和HEK 293细胞的实验结果使我们更好地理解了亚基组装、细胞内信号传导过程和跨膜结构域如何调节该受体对酒精的敏感性。 在本申请中，PI将通过学习用于表达脑神经元中乙醇敏感性改变的突变型NMDA受体亚基的技术，并评估分子生物学，电生理学和动物行为的特定领域，扩展这些发现的相关性。 具体而言，PI将：1）学习产生和使用基于病毒的基因载体将具有改变的乙醇敏感性的NMDA受体亚单位递送到培养物和体内生长的神经元中所需的技术; 2）学习用于产生表达具有改变的乙醇敏感性的突变NMDA受体亚单位的遗传修饰动物的方法; 3）学习制备和记录从表达具有改变的乙醇敏感性的突变NMDA受体的动物分离的脑切片的离子通道电流的方法; 4）学习评估酒精在表达突变型NMDA受体的动物中的神经行为作用所需的技术，这些动物具有改变的酒精敏感性。 这种培训将在一系列课程中进行，这些课程由各种具有技术专长的顾问监督。 成功完成本培训计划将大大提高PI的研究专业知识和实现其研究目标的能力。