Characterization of Cardiac Purinoceptors

性状（申请人逐字描述）：三磷酸腺苷 (ATP)在影响细胞活性方面是不寻常的， 细胞内和细胞外区室。ATP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， 在许多生物合成反应中磷酸盐的重要来源。细胞外ATP 通过激活膜结合的P2嘌呤受体调节细胞兴奋性。一 该家族的分支P2 X受体本身是一类 配体门控离子通道，其引导阳离子流过细胞 各种组织的表面膜。阳离子传导发生在 整合离子通道开放是由于激动剂占据 胞外结合位点。然而，门控的分子机制和 选择性仍然是一个谜，部分原因是由于一个不完整的映射， 受体-通道复合物的功能域。此外，这些角色 蛋白质在控制天然组织的细胞兴奋性方面的作用很差， 定义了本提案中概述的实验旨在解决 这些缺陷使用了重组嘌呤能受体， 人胚肾细胞与急性分离大鼠天然受体 心房肌第一个具体目标是绘制 P2 X2受体的结构和功能。我们建议鉴定氨基酸 这有助于选择性过滤器和通道门使用 包含性定点诱变方法，其考虑了假定的 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探讨了这些领域的贡献 新发现的通道孔隙的“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 渗透途径使用的小的一价和二价阳离子在 “收缩”孔隙模式。同样，定点突变将用于 确定扩张的二级和三级结构的差异， 狭窄的渠道。第三个具体的目标是描述当地人的特点， 大鼠心房肌的嘌呤能电流。虽然ATP已知可以 通过激活P2 X受体家族的一个未知成员， 关于ATP对哺乳动物心房的作用知之甚少。我们的初步 数据强烈提示大鼠心房表达功能性P2 X受体， 独特.更完整的生理学和药理学特征， 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.