G PROTEIN NULL MUTATION AND CARDIAC RECEPTOR SIGNALING

G 蛋白无效突变和心脏受体信号传导

• 批准号: 2713926
• 负责人: MARGARET O SOWELL
• 金额: $ 9.2万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-01-01 至 2001-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2713926
• 关键词: CHO cells; G protein; acetylcholine; adenosine; biological signal transduction; embryonic stem cell; gene deletion mutation; gene targeting; heart cell; heart contraction; heart rate; muscarinic receptor; northern blottings; potassium channel; purinergic receptor; receptor coupling; southern blotting; voltage /patch clamp; western blottings

Numerous reports have documented changes in the amounts or functional activity of G proteins in cardiac pathology and with aging. Given the potential clinical impact of these observations it is critical that we gain a clear understanding of the normal signal transduction pathways. The studies outlined in this proposal will provide a framework for understanding what clinical significance these changes may have and may help in devising clinically useful interventions. The goal of this proposal is to use targeted mutagenesis of specific G protein genes as a tool to investigate cardiac muscarinic and adenosine receptor-G protein signaling mechanisms. Acetylcholine and adenosine are two important inhibitory modulators of cardiac performance and may have cardioprotective effects during periods of ischemia. Using mouse embryonic stem (ES) cells a panel of mutant lines will be generated in which one or more of the G protein subunits (alpha, beta, or gamma) is inactivated. The effects of these inactivations on receptor signaling will then be evaluated using in vitro differentiated ES cells as a model for cardiocytes. Our specific goals are to identify the G protein subunits required by each receptor for signaling to a common effector and by a single receptor for signaling to two different effectors (the atrial-type inwardly rectifying K+ channel and the ATP sensitive K+ channel). Chronotropic responses will also be monitored as an indicator of overall receptor signaling efficiency and will provide a physiological correlate for our single channel studies. Although I have had some training in cell physiology and metabolism with my PhD, to become an independent investigator, it is critical that I obtain extensive training in genetic and molecular techniques. After a five year hiatus during which I completed clinical training, I joined Dr. Richard Mortensen's laboratory to gain hands-on experience with molecular biology. To complete the studies described in this research plan, training in electrophysiology (under the guidance of Dr. Peter Vassilev) is also required. I have a very strong advisory committee of three experienced senior scientists, Dr. Gordon Williams, Dr. Christine Seidman, and Dr. Eva Neer, who will provide an invaluable resource for my scientific and personal career development. Long term I plan to use information gained from the studies outlined in this proposal to further investigate the role that G proteins play in cardiac disease and to characterize factors (metabolic, hormonal, and pharmacologic) that regulate expression and function of theses proteins. The ultimate objective is to be able to predictably and specifically modulate the functions of G proteins in a clinically beneficial manner.

许多报告记录了金额或功能的变化 G蛋白的活性在心脏病理和衰老中的作用。鉴于 这些观察结果的潜在临床影响至关重要， 获得对正常信号转导途径的清晰理解。 本提案中概述的研究将提供一个框架， 了解这些变化的临床意义， 帮助设计临床上有用的干预措施。 本提案的目的是利用特异性G 蛋白质基因作为研究心脏毒蕈碱和腺苷的工具 受体-G蛋白信号传导机制。 乙酰胆碱和腺苷是 两种重要的心脏功能抑制调节剂， 在缺血期间的心脏保护作用。使用小鼠 胚胎干（ES）细胞中将产生一组突变株系， 其中一个或多个G蛋白亚基（α，β或γ）是 灭活这些失活对受体信号传导的影响 然后将使用体外分化的ES细胞作为模型进行评估 对于心肌细胞。我们的具体目标是确定G蛋白 每个受体所需的用于向共同效应物发出信号的亚基， 通过单个受体向两个不同的效应器（ 心房型内向整流钾通道与ATP敏感性钾通道 频道）。也将监测变时性反应作为指标 的整体受体信号传导效率，并将提供一个生理 与我们的单通道研究相关。 虽然我在细胞生理学和新陈代谢方面受过一些训练， 我的博士学位，成为一个独立的调查员，这是至关重要的，我 在遗传和分子技术方面获得广泛的培训。后 在我完成临床培训的五年中断期间，我加入了Dr. 理查德·莫滕森的实验室，以获得实际经验的分子 生物学为了完成本研究计划中描述的研究， 电生理学培训（在Peter Vassilev博士的指导下） 也是必需的。我有一个非常强大的三人顾问委员会 经验丰富的资深科学家，戈登威廉姆斯博士，克莉丝汀博士 塞德曼和伊娃尼尔博士，谁将提供一个宝贵的资源， 我的科学和个人职业发展。长期计划使用 从本提案中概述的研究中获得的信息， 研究G蛋白在心脏病中的作用， 表征因素（代谢、激素和药理学）， 调节这些蛋白的表达和功能。最终 目的是能够可预测地和特异性地调节 以临床有益的方式发挥G蛋白的功能。