Structure/Function Studies on the Mechanisms of Purinergic Receptor Activation and Antagonism

项目摘要 嘌呤能（P2 X）受体是由ATP激活的三聚体非选择性阳离子通道，其在细胞内起重要作用。 在心血管、神经和免疫系统中的作用。尽管它们在人体生理学中起着重要作用， 作为治疗剂的潜在靶点，P2 X受体拮抗作用的分子机制尚不清楚， 特别是对于几乎一无所知的非竞争性拮抗剂。P2 X受体的研究 由于缺乏作为选择性高亲和力激动剂的小分子， 针对各种受体亚型的拮抗剂。最近，申请人公布了原子分辨率 在apo/静息状态、激动剂结合/开孔状态、激动剂结合/开孔状态下的人P2 X3（hP 2X 3）结构 结合/闭孔/脱敏状态和两个竞争性拮抗剂结合状态，确定了一种新的 他假设胞质结构域在受体激活中起着关键和独特的结构作用， 脱敏这笔赠款的目的是为了加强这项工作，以牢固地建立 P2 X受体拮抗作用的原理，确定参与的关键细胞质残基的结构作用， P2 X受体的激活和脱敏，并确定新的小分子竞争性和非竞争性 针对同源三聚体P2 X1和P2 X3受体亚型和异源三聚体P2 X2，3受体的拮抗剂， 利用配体结合分析和电生理学研究功能和X射线晶体学和冷冻 电子显微镜研究结构。这项研究将由受过良好培训的申请人进行 在膜蛋白生物化学和强大的出版记录。本提案的培训将在 俄勒冈州健康与科学大学的Vollum研究所在Eric Gouaux博士的指导下， 离子通道和转运蛋白结构生物学的领导者，他以前曾在许多 博士后学员独立。为使申请人为成功过渡到独立做好准备， 博士Gouaux将监督候选人在低温电子显微镜方面的结构生物学培训，并继续 X射线晶体学中的生长除了Gouaux博士之外，一个由知名研究人员组成的研究所也支持 申请人具有膜蛋白生物化学、配体-受体相互作用/结合试验方面的专业知识， 离子通道的电生理学这种培训对于候选人成为一名 一位独立的研究者专注于研究嘌呤能受体的结构和功能， 开发用于治疗心血管疾病如心绞痛，高血压， 和血小板聚集。

PROJECT SUMMARY Purinergic (P2X) receptors are trimeric, non-selective cation channels activated by ATP that play important roles in cardiovascular, neuronal and immune systems. Despite their central function in human physiology and as potential targets of therapeutic agents, the molecular mechanisms for P2X receptor antagonism are unclear, especially for non-competitive antagonists where almost nothing is known. The study of P2X receptors has been handicapped by a paucity of small molecules that serve as selective high-affinity agonists and antagonists against the various receptor subtypes. Very recently, the applicant published atomic resolution structures of human P2X3 (hP2X3) in an apo/resting state, an agonist-bound/open-pore state, an agonist- bound/closed-pore/desensitized state and two competitive antagonist-bound states, identifying a novel cytoplasmic domain that he hypothesizes plays a pivotal and unique structural role in receptor activation and desensitization. The aims of this grant are designed to build off that work in order to firmly establish the principles of P2X receptor antagonism, determine the structural role of key cytoplasmic residues involved in P2X receptor activation and desensitization, and identify novel small molecule competitive and non-competitive antagonists directed at homo-trimeric P2X1 and P2X3 receptor subtypes and hetero-trimeric P2X2,3 receptors, utilizing ligand binding assays and electrophysiology to study function and X-ray crystallography and cryo electron microscopy to study structure. This research will be carried out by an applicant with excellent training in membrane protein biochemistry and a strong publication record. The training for this proposal will occur at the Vollum Institute of Oregon Health and Science University under the mentorship of Dr. Eric Gouaux, a world leader in the structural biology of ion channels and transporters who has previously transitioned numerous post-doctoral trainees to independence. To prepare the applicant for a successful transition to independence, Dr. Gouaux will oversee the candidate's structural biology training in cryo electron microscopy and continued growth in X-ray crystallography. In addition to Dr. Gouaux, an institute of established investigators supports the applicant with expertise in membrane protein biochemistry, ligand-receptor interactions/binding assays, and the electrophysiology of ion channels. This training will be essential for the candidate to become an independent investigator focused on studying the structure and function of purinergic receptors in order to develop pharmacological agents for the treatment of cardiovascular conditions such as angina, hypertension, and platelet aggregation.