Sites of Ethanol Action in Purinergic P2X4 Receptors

嘌呤能 P2X4 受体中乙醇的作用位点

• 批准号: 7923720
• 负责人: LIANA ASATRYAN
• 金额: $ 12.26万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-20 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7923720
• 关键词: Alanine; Alcohol consumption; Alcohols; Amino Acid Substitution; Amino Acids; Area; Behavioral; Cells; Chemosensitization; Electrodes; Electrophysiology (science); Ethanol; Faculty; Family; Gated Ion Channel; Goals; Hand; Investigation; Ion Channel; Knockout Mice; Laboratories; Lead; Ligands; Mammalian Cell; Mediating; Molecular; Mutagenesis; Neuraxis; Neurons; Oocytes; Pathway interactions; Pharmacy Schools; Play; Positioning Attribute; Post-Translational Protein Processing; Process; Recombinants; Research; Research Personnel; Role; Scanning; Site; Site-Directed Mutagenesis; Staging; System; Techniques; Testing; Training; Transmembrane Domain; Work; Xenopus; Xenopus oocyte; alcohol effect; alcohol research; alcohol sensitivity; alcoholism therapy; career; design; extracellular; member; mutant; new therapeutic target; patch clamp; receptor; receptor sensitivity; research study; voltage clamp

DESCRIPTION (provided by applicant): P2X4 receptor (P2X4R) is a member of a family of ligand-gated ion channels that are activated by extracellular ATP. P2X4Rs are widely distributed throughout the central nervous system and are inhibited by ethanol. Building evidence suggests that ethanol inhibition may play a role in mediating and/or modulating some of the cellular and behavioral effects of ethanol. However, there is a paucity of information regarding the sites of ethanol action on P2XRs. Recent work in our laboratory, utilizing a chimeric strategy in P2X2R/P2X3Rs, suggests that regions in P2X3R ectodomain near the transmembrane (TM) domains and the TM1 domain itself may represent important targets for ethanol. My K01 proposal extends this line of investigation to P2X4Rs by testing the hypothesis that the ectodomain region and the TM1 domain of P2X4Rs contain sites of ethanol action. The primary goals of this project are: 1) Provide extensive hands-on training for myself in cellular, molecular and electrophysiological techniques that will be necessary in order to take the next step towards becoming an independent alcohol researcher; 2) To identify important sites of ethanol action in P2X4Rs. Results from the proposed studies will set the stage for new directions of independent investigation for myself in alcohol/purinergic research. To accomplish these goals two Specific Aims are proposed. Aim 1 investigates the role of the ectodomain and TM1 domain of P2X4Rs in ethanol action using advanced electrophysiological techniques and recombinant, cellular expression systems. In these studies I will utilize alanine scanning mutagenesis of the indicated regions of P2X4Rs to identify key amino acids involved in ethanol action. Aim 2 investigates the effects of ethanol on P2X4Rs and P2X4-like receptors in dissociated neurons. These later studies will utilize P2X4R-transfected neurons as well as neurons from control and P2X4R-knockout mice. Results of proposed studies will identify ethanol targets in endogenous and recombinant P2X4Rs. The long-term goal of the proposed work is to determine the role of P2X4Rs in mediating and/or modulating behavioral effects of ethanol and where warranted to identify novel therapeutic targets for the treatment of alcoholism. My K01 proposal represents advanced experimental training and new directions in my scientific career and provides me with an opportunity to become an independent alcohol researcher working towards a faculty position at USC School of Pharmacy.

描述（由申请人提供）：P2X4受体（P2X4R）是由细胞外ATP激活的配体门控离子通道家族的成员。 P2X4R广泛分布在整个中枢神经系统中，并被乙醇抑制。建筑证据表明，乙醇抑制作用可能在介导和/或调节乙醇的某些细胞和行为作用中起作用。但是，关于P2XRS乙醇作用的位点的信息很少。在我们的实验室中，利用P2X2R/P2X3R中的嵌合策略的最新工作表明，跨膜（TM）域（TM）域和TM1域本身附近的P2X3R外生域的区域可能代表乙醇的重要目标。我的K01提案通过测试了p2x4rs的胞外域区域和TM1结构域包含乙醇作用位点的假设，将此研究线扩展到P2X4R。该项目的主要目标是：1）在细胞，分子和电生理技术方面为自己提供广泛的动手培训，这是为了迈出独立的酒精研究人员的下一步而必要的； 2）确定P2X4R中乙醇作用的重要位点。拟议的研究的结果将为我在酒精/嘌呤能研究中为自己独立研究的新方向奠定阶段。为了实现这些目标，提出了两个具体目标。 AIM 1使用先进的电生理技术和重组，细胞表达系统研究了P2X4RS的胞外域和TM1结构域在乙醇作用中的作用。在这些研究中，我将利用P2X4RS指定区域的丙氨酸扫描诱变来鉴定参与乙醇作用的关键氨基酸。 AIM 2研究了乙醇对解离神经元中P2X4R和P2X4样受体的影响。这些后来的研究将利用P2X4R转染的神经元以及对照和P2X4R-敲除小鼠的神经元。拟议的研究结果将确定内源性和重组P2X4R中的乙醇靶标。拟议工作的长期目标是确定P2X4R在介导和/或调节乙醇的行为效应中的作用，并有必要在其中确定新颖的治疗靶标在治疗酒精中毒的治疗方面。我的K01提案代表了我的科学生涯中的高级实验培训和新的方向，并为我提供了成为一名独立的酒精研究人员，致力于在USC药学学院担任教职员工。