Characterization of Cardiac Purinoceptors

心脏嘌呤受体的表征

• 批准号: 6333611
• 负责人: TERRANCE M EGAN
• 金额: $ 24.94万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6333611
• 关键词: adenosine triphosphate; laboratory rat; membrane channels; membrane potentials; myocardium; protein structure function; purinergic receptor; receptor binding; receptor expression; recombinant proteins; site directed mutagenesis; tissue /cell culture

DESCRIPTION (the applicant's description verbatim): Adenosine triphosphate (ATP) is unusual in its ability to influence cell activity from both the intracellular and extracellular compartments. Intracellular hydrolysis of ATP to adenosine 5'-diphosphate and inorganic phosphate provides the energy needed to power a range of energetically unfavorable chemical reactions and is an important source of phosphate in many biosynthetic reactions. Extracellular ATP modulates cell excitability by activating membrane-bound P2 purinoceptors. One branch of this family, the P2X receptors, are themselves a class of ligand-gated ionic channels that conduct the flow of cations across the cell surface membranes of a variety of tissue. Cationic conduction occurs when the integrant ion channel opens as a result of agonist occupation of an extracellular binding site. However, the molecular mechanisms of gating and selectivity remain a mystery due in part to an incomplete mapping of the functional domains of the receptor-channel complex. Further, the role these proteins play in control of cell excitability of native tissues is poorly defined. The experiments outlined in this proposal are designed to address these deficiencies using both recombinant purinergic receptors expressed in human embryonic kidney cells and native receptors of acutely-dissociated rat atrial muscle. The first specific aim is to map the relationship between structure and function of the P2X2 receptor. We propose to identify amino acid that contribute to the selectivity filter and the channel gate using an inclusive site-directed mutagenesis approach that considers both putative pore-forming domains. Our preliminary data demonstrate this approach will be successful. The second specific aim explores the contribution of these domains to the newly discovered "dilated" mode of the channel pore. We hypothesize that the large cations that permeate the dilated channel traverse the same permeation pathway used by small mono- and divalent cations in the "constricted" pore mode. Again, site-directed mutagenesis will be used to identify differences in the secondary and tertiary structure of the dilated and constricted channels. The third specific aim is to characterize the native purinergic current of rat atrial muscle. Although ATP is known to depolarize frog atrial muscle by activating an unknown member of the P2X receptor family, little is known about the effect of ATP on mammalian atria. Our preliminary data strongly suggest that rat atria express a functional P2X receptor that is unique. A more complete characterization of the physiology and pharmacology of this receptor will help define the role of ATP in the pharmacology, physiology, and pathophysiology o mammalian cardiac muscle.

描述（申请人的逐字描述）：三磷酸腺苷 (ATP) 的不同寻常之处在于它能够从以下两个方面影响细胞活动： 细胞内和细胞外区室。 ATP 的细胞内水解 腺苷5'-二磷酸和无机磷酸提供所需的能量 为一系列能量上不利的化学反应提供动力，并且是一种 许多生物合成反应中磷酸盐的重要来源。细胞外ATP 通过激活膜结合 P2 嘌呤受体来调节细胞兴奋性。一 这个家族的一个分支，P2X 受体，本身就是一类 引导阳离子穿过细胞流动的配体门控离子通道 各种组织的表面膜。当发生阳离子传导时 整合离子通道由于激动剂占据而打开 细胞外结合位点。然而，门控和 选择性仍然是一个谜，部分原因是不完整的映射 受体通道复合物的功能域。此外，这些作用 蛋白质在控制天然组织的细胞兴奋性方面的作用很差 定义的。本提案中概述的实验旨在解决 这些缺陷使用表达的重组嘌呤能受体 人胚胎肾细胞和急性解离大鼠的天然受体 心房肌。第一个具体目标是绘制之间的关系 P2X2 受体的结构和功能。我们建议鉴定氨基酸 有助于选择性滤波器和通道门使用 包容性定点诱变方法，考虑了假定的 成孔域。我们的初步数据表明这种方法将 成功的。第二个具体目标探讨这些领域的贡献 到新发现的通道孔的“扩张”模式。我们假设 渗透扩张通道的大阳离子穿过相同的通道 小单价和二价阳离子使用的渗透途径 “收缩”孔隙模式。同样，定点诱变将用于 识别扩张和扩张的二级和三级结构的差异 渠道狭窄。第三个具体目标是刻画本地人的特征 大鼠心房肌的嘌呤能电流。尽管已知 ATP 具有去极化作用 通过激活 P2X 受体家族的未知成员来青蛙心房肌， 关于 ATP 对哺乳动物心房的影响知之甚少。我们的初步 数据强烈表明大鼠心房表达功能性 P2X 受体 独特的。更完整的生理学和药理学表征 该受体将有助于定义 ATP 在药理学、生理学、 和哺乳动物心肌的病理生理学。