G PROTEIN NULL MUTATION AND CARDIAC RECEPTOR SIGNALING

许多报告记录了金额或功能的变化 G蛋白在心脏病理中的活性及其与增龄的关系给定 这些观察结果的潜在临床影响至关重要的是我们 对正常的信号转导途径有一个清晰的了解。 本提案中概述的研究将提供一个框架， 了解这些变化可能具有的临床意义 帮助设计临床上有用的干预措施。 这项提议的目标是使用特定G基因的定向突变 蛋白质基因作为研究心肌毒副反应和腺苷的工具 受体-G蛋白信号转导机制。乙酰胆碱和腺苷是 两种重要的心功能抑制调节剂，可能具有 缺血期间的心脏保护作用。使用鼠标 胚胎干细胞(ES)一组突变株将在 哪一个或多个G蛋白亚基(α、β或伽马)是 已停用。这些失活对受体信号转导的影响 然后将使用体外分化的ES细胞作为模型进行评估 对心肌细胞来说。我们的具体目标是确定G蛋白 每个受体向共同效应器发送信号所需的亚基 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敏感性钾离子 频道)。变时性反应也将作为一项指标进行监测 整体受体信号转导效率，并将提供一个生理 与我们的单渠道研究相关。 虽然我曾接受过一些细胞生理学和新陈代谢方面的培训 我的博士学位，要成为一名独立的调查员，关键是我 获得遗传和分子技术方面的广泛培训。在.之后 在我完成临床培训的五年时间里，我加入了Dr。 理查德·莫滕森的实验室将获得分子实践经验 生物学。为了完成本研究计划中描述的研究， 电生理学培训(由彼得·瓦西列夫博士指导) 也是必需的。我有一个非常强大的咨询委员会，由三人组成 经验丰富的资深科学家戈登·威廉姆斯博士、克里斯汀博士 Seidman和Eva Neer博士，他们将为 我的科学和个人事业发展。从长远来看，我计划使用 从本提案概述的研究中获得的信息，以进一步 研究G蛋白在心脏病中所起的作用 描述以下因素(代谢、荷尔蒙和药物) 调节这些蛋白质的表达和功能。终极的 目标是能够可预测地和特定地调节 G蛋白在临床上有益的功能。

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.