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

描述（由申请人提供）：本申请提出了一个职业发展计划博士克里斯托弗O。兰达克发展成为一个独立的物理学家，科学家和学术教师和导师。该提案的核心是强化实验室培训经验，以培养蛋白质化学和膜蛋白生物化学方面的科学技能和理解。囊性纤维化跨膜传导调节因子（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，即腺苷5'-三磷酸（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。许多先前的研究集中在CFTR的ATP酶活性及其如何支配CFTR门控。最近的研究表明，CFTR也与腺苷5'-单磷酸（AMP）相互作用，腺苷酸激酶活性（ATP：AMP磷酸转移）可以门控CFTR。本研究的目的是确定CFTR的AMP结合和腺苷酸激酶活性的关键机制和关键氨基酸残基。利用叠氮核苷酸标记技术和CFTR膜片钳技术，将检验AMP和ATP与CFTR协同作用的假设，以及AMP阻止ATP水解并诱导ATP：AMP磷酸转移的假设。这些结果有望阐明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;还有望确定参与AMP与CFTR结合的关键残基。该计划将为Randak博士提供正式的职业指导和指导。迈克尔·J·威尔士博士将承担导师的责任，杰弗里·C. Murray，Paul B.小麦克雷凯文·P·坎贝尔将担任顾问。其他目标是增加Randak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资金做好准备。爱荷华州大学将为Randak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。这样的化合物可用于治疗与太少（囊性纤维化）或太多（分泌性腹泻）CFTR氯电流相关的疾病。由于CFTR是一种ABC转运蛋白，这一知识也可能影响其他ABC转运蛋白相关疾病的治疗。 (End摘要）

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)