Mechanisms and Binding-Sites in Adenylate Kinase-Dependent CFTR Gating

描述（由申请人提供）：本申请为 Christoph O. Randak 博士提出职业发展计划，以发展成为一名独立的医师科学家、学术教师和导师。该提案的核心是密集的实验室培训经验，以培养科学技能和对蛋白质化学和膜蛋白生物化学的理解。囊性纤维化跨膜电导调节器 (CFTR) 是一种受调节的氯离子通道，是膜蛋白最大家族之一的代表，即 5'-三磷酸腺苷 (ATP) 结合盒 (ABC) 转运蛋白。编码 CFTR 的基因突变会导致囊性纤维化 (CF)。了解 CFTR 通道门控的分子机制将有助于开发治疗和控制 CF 的方法。之前的许多研究都集中在 CFTR 的 ATP 酶活性及其如何控制 CFTR 门控上。最近的研究表明，CFTR 还与腺苷 5'-单磷酸 (AMP) 相互作用，并且腺苷酸激酶活性（ATP:AMP 磷酸转移）可以控制 CFTR。本研究计划的目的是确定参与 AMP 结合和 CFTR 腺苷酸激酶活性的关键机制和关键氨基酸残基。使用叠氮核苷酸和 CFTR 膜片钳标记技术，将测试以下假设：AMP 和 ATP 与 CFTR 协同相互作用，以及 AMP 防止 ATP 水解并诱导 ATP:AMP 磷酸转移。研究结果有望阐明 ATP 和 AMP 如何相互影响它们与 CFTR 的相互作用，以及 AMP 如何影响 CFTR 的酶活性；它还有望鉴定参与 AMP 与 CFTR 结合的关键残基。该计划将为兰达克博士提供正式的职业指导和指导。 Michael J. Welsh 博士将担任导师，Drs. Jeffrey C. Murray、Paul B. McCray Jr. 和 Kevin P. Campbell 将担任顾问。其他目标是增加兰达克博士的教学和指导经验，指导他建立研究实验室，提高他的科学演讲和写作技能，帮助他扩大与其他科学家的联系和合作，并为他申请独立研究经费做好准备。爱荷华大学将为兰达克博士的科学发展和学术成功提供最先进的研究环境。相关性（参见说明）：这些研究的知识将为开发 CFTR 通道功能的激活剂和抑制剂提供框架。此类化合物可用于治疗与CFTR氯化物电流过少（囊性纤维化）或过多（分泌性腹泻）相关的疾病。由于 CFTR 是 ABC 转运蛋白，因此这一知识也可能影响其他 ABC 转运蛋白相关疾病的治疗。 （摘要完）

DESCRIPTION (provided by applicant): This application proposes a career development program for Dr. Christoph O. Randak to develop into an independent physician-scientist and academic teacher and mentor. The heart of the proposal is an intensive laboratory training experience to develop scientific skills and understanding in protein chemistry and biochemistry of membrane proteins. The cystic fibrosis transmembrane conductance regulator (CFTR), a regulated chloride channel, is a representative of one of the largest families of membrane proteins, the adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporters. Mutations in the gene encoding CFTR cause cystic fibrosis (CF). Understanding the molecular mechanisms of CFTR channel gating will facilitate the development of means to cure and control CF. Many previous studies have focused on CFTR's ATPase activity and how it governs CFTR gating. Recent work has shown that CFTR also interacts with adenosine 5'-monophosphate (AMP) and that adenylate kinase activity (ATP:AMP phosphotransfer) can gate CFTR. The objective of this research proposal is to identify key mechanisms and key amino acid residues involved in AMP-binding and adenylate kinase activity of CFTR. Using labeling techniques with azido-nucleotides and CFTR patch-clamping, the hypothesis will be tested that AMP and ATP interact cooperatively with CFTR, and that AMP prevents ATP hydrolysis and induces ATP:AMP phosphotr-ansfer. The results are expected to elucidate how ATP and AMP mutually influence their interaction with CFTR, and how AMP influences CFTR's enzymatic activity; it is also expected to identify key residues involved in AMP-binding to CFTR. The program will provide Dr. Randak with formal career mentoring and guidance. Dr. Michael J. Welsh will assume responsibility as mentor and Drs. Jeffrey C. Murray, Paul B. McCray Jr., and Kevin P. Campbell will be advisors. Additional objectives are to increase Dr. Randak's teaching and mentoring experience, to guide him in establishing a research laboratory, to increase his skills in scientific speaking and writing, to help him expand his contacts and collaborations with other scientists, and to prepare him to apply for independent research funding. The University of Iowa will provide a state-of-art research environment dedicated to Dr. Randak's scientific development and academic success. RELEVANCE (See instructions): Knowledge from these studies will provide a framework to develop activators and inhibitors of CFTR channel function. Such compounds may be useful for the treatment of diseases associated with too little (cystic fibrosis) or too much (secretory diarrhea) CFTR chloride currents. Because CFTR is an ABC transporter, this knowledge may also impact treatments of other ABC transporter-related diseases. (End of Abstract)