REGULATION OF MAMMALIAN CARDIAC MUSCARINIC RECEPTORS

哺乳动物心肌毒蕈碱受体的调节

基本信息

批准号：
2638097
负责人：
Neil Marc Nathanson
金额：
$ 24.03万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-04-01 至 2002-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2638097
关键词：
G protein acetylcholine arrestins gene targeting genetically modified animals heart function laboratory mouse luciferin monooxygenase muscarinic receptor protein kinase protein sequence receptor binding receptor coupling receptor expression site directed mutagenesis tissue /cell culture transfection /expression vector

项目摘要

DESCRIPTION (Adapted from Applicant's Abstract): The overall goal of this research is to elucidate the mechanisms involved in the regulation and function of muscarinic acetylcholine receptors (mAChR) in the mammalian heart. Activation of the mAChR causes a decrease in the rate and force of contraction, and regulates the activities of adenylyl cyclase, phospholipase C, and potassium and calcium channels. While most cardiac mAChR are of the m2 subtype, a wide variety of pharmacological, physiological, immunological, biochemical, and molecular biological evidence indicates that the m1 receptor is also present at very low levels in the heart. The function and number of mAChR can be decreased by continued exposure of agonist. The PI has recently demonstrated that the beta-adrenergic receptor kinase and beta-arrestin synergistically regulate signal transduction by the m2 receptor in transfected cells. This proposal will test the hypothesis that the m2 and m4 receptors exhibit different selectivities for regulation by subtypes of G-protein receptor kinases and arrestins, and determine the functional consequences of elimination of the phosphorylation sites on mAChR function and regulation. Short-term agonist exposure also causes sequestration of receptors from the cell surface. Previous results indicate that the m1 and m2 receptors use distinct cellular mechanisms for agonist-induced internalization. This proposal will test the hypothesis that a portion of the m2 receptor in the sixth transmembrane domain and the third cytoplasmic domain (i3)form an contiguous sequestration domain. This proposal will identify specific residues and determine the effects of lack of sequestration on the regulation of muscarinic responsiveness. This proposal will also alter expression and introduce mutations into mAChR in the heart in vivo to provide unique information on the regulation of mAChR signaling in the heart. This proposal will use m1 knockout mice generated by this laboratory to determine the role of the m1 receptor in the physiological actions of acetylcholine and the regulation of mAChR expression in the heart. This project will determine the effect of disruption of the m2 receptor on cardiac function, responses to ACh, and expression of other mAChR subtypes, and will introduce mutations into the m2 receptor in mice to test the role of phosphorylation and sequestration on receptor function and signaling in the heart in vivo. This research will provide valuable new information on the basic mechanisms regulating the expression and function of mAChR in the heart. In addition, this research may aid in understanding the etiology of a variety of cardiac abnormalities.

描述（改编自申请人的摘要）：本项目的总体目标研究的目的是阐明调节和调节所涉及的机制哺乳动物毒蕈碱乙酰胆碱受体 (mAChR) 的功能心。 mAChR 的激活会导致速率和作用力降低收缩，并调节腺苷酸环化酶、磷脂酶的活性 C、钾和钙通道。虽然大多数心脏 mAChR 属于 m2亚型，种类繁多，药理学、生理学、免疫学、生化和分子生物学证据表明 m1 受体在心脏中的含量也非常低。功能和 mAChR 的数量可以通过持续暴露激动剂而减少。 PI 最近证明，β-肾上腺素受体激酶和 beta-arrestin 协同调节 m2 的信号转导转染细胞中的受体。该提案将检验以下假设： m2 和 m4 受体表现出不同的调节选择性 G 蛋白受体激酶和抑制蛋白的亚型，并确定消除 mAChR 磷酸化位点的功能后果功能和调节。短期激动剂暴露也会导致受体与受体隔离细胞表面。先前的结果表明 m1 和 m2 受体使用激动剂诱导内化的独特细胞机制。这该提案将检验以下假设：m2 受体的一部分第六跨膜结构域和第三胞质结构域 (i3) 形成连续的隔离域。该提案将确定具体残留物并确定缺乏封存对毒蕈碱反应性的调节。该提案还将改变 mAChR 的表达并引入突变在心脏体内提供有关调节的独特信息心脏中的 mAChR 信号传导。本提案将使用 m1 基因敲除小鼠该实验室产生的用于确定 m1 受体在乙酰胆碱的生理作用和mAChR的调节表达在心里。该项目将决定效果 m2 受体的破坏对心脏功能、ACh 反应和其他 mAChR 亚型的表达，并将突变引入 m2 小鼠受体以测试磷酸化和隔离的作用心脏体内受体功能和信号传导。这项研究将为基本机制提供有价值的新信息调节心脏中 mAChR 的表达和功能。此外，这项研究可能有助于了解多种心脏病的病因异常。