Characterization of Cardiac Purinoceptors

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两个方面影响细胞活动 细胞内和细胞外的隔间。三磷酸腺苷的胞内水解 向腺苷5‘-二磷酸和无机磷提供所需的能量 为一系列能量不利的化学反应提供动力，这是一种 许多生物合成反应中磷酸盐的重要来源。胞外三磷酸腺苷 通过激活膜上的P2嘌呤受体来调节细胞的兴奋性。一 这个家族的分支，即P2X受体，本身就是一类 配基门控离子通道，引导阳离子在细胞内流动 各种组织的表面膜。阳离子传导发生在 整合离子通道开放作为激动剂占领的结果 胞外结合部位。然而，门控和门控的分子机制 选择性仍然是一个谜，部分原因是对 受体-通道复合体的功能域。此外，这些角色 蛋白质在控制天然组织细胞兴奋性中的作用很差 已定义。本提案中概述的实验旨在解决 这些缺陷使用了两种表达在 人胚胎肾细胞与急性分离大鼠的天然受体 心房肌。第一个具体目标是绘制出 P2X2受体的结构和功能。我们建议鉴定氨基酸 用于选择性过滤器和通道门的 一种包容性定点突变方法，它考虑了假设的 成孔域。我们的初步数据表明，这种方法将是 成功。第二个具体目标是探索这些领域的贡献 到了新发现的通道孔的“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 一价和二价小阳离子在土壤中的渗透途径 “收缩”的毛孔模式。再一次，定点突变将被用于 确定扩张型和扩张型的二级结构和三级结构的差异 狭窄的航道。第三个具体目标是描述原住民的特征 大鼠心房肌的嘌呤能电流。尽管已知三磷酸腺苷能使人去极化 通过激活一个未知的P2X受体家族成员来激活青蛙心房肌， 关于三磷酸腺苷对哺乳动物心房的影响，人们知之甚少。我们的预赛 数据有力地表明，大鼠心房表达一种功能性的P2X受体 独一无二。更完整的生理和药理学特征 这种受体将有助于确定ATP在药理学、生理学、 和哺乳动物心肌的病理生理学。

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.