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

腺苷酸激酶依赖性 CFTR 门控的机制和结合位点

• 批准号: 8308495
• 负责人: Christoph Oskar Randak
• 金额: $ 12.74万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-13 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8308495
• 关键词: ATP phosphohydrolase; ATP-Binding Cassette Transporters; Address; Adenosine; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Amino Acids; Arts; Binding; Binding Sites; Biochemical; Biological; Carrier Proteins; Chloride Channels; Chloride Ion; Chlorides; Collaborations; Coupled; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Data; Dependency; Development; Diarrhea; Diphosphates; Disease; Educational process of instructing; Electrophysiology (science); Elements; Environment; Family; Funding; Genes; Goals; Heart; Hydrolysis; Instruction; Iowa; Knowledge; Label; Laboratories; Laboratory Research; Length; Membrane; Membrane Proteins; Mentors; Methods; Molecular; Mutation; Nucleotides; Outcome; Physicians; Physiological; Principal Investigator; Protein Biochemistry; Protein Chemistry; Protein Family; Proteins; Publishing; Quality of life; Recombinants; Research; Research Personnel; Research Proposals; Role; Scientist; Site; Structure-Activity Relationship; Techniques; Testing; Training; Universities; Work; Writing; abstracting; adenylate kinase; base; career; career development; cystic fibrosis patients; experience; human disease; improved; in vivo; inhibitor/antagonist; innovation; inorganic phosphate; kinase inhibitor; member; membrane activity; novel; patch clamp; pediatric department; prevent; programs; protein function; reconstitution; skills; success; teacher; tripolyphosphate

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)

描述(由申请者提供)：此申请表建议克里斯托弗·O·兰达克博士发展成为一名独立的内科科学家、学术教师和导师的职业发展计划。该提案的核心是强化实验室培训经验，以发展蛋白质化学和膜蛋白质生物化学方面的科学技能和理解。囊性纤维化跨膜电导调节器是一种受调节的氯离子通道，是最大的膜蛋白家族之一，是三磷酸腺苷结合盒转运体(ABC)的代表。CFTR编码基因的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。以前的许多研究都集中在cftr的ATPase活性以及它如何控制cftr门控。最近的研究表明，CFTR也可以与腺苷5‘-一磷酸(AMP)相互作用，并且腺苷酸激酶活性(ATP：AMP磷酸转移)可以门控CFTR。本研究的目的是确定cftr结合AMP和腺苷酸激酶活性的关键机制和关键氨基酸残基。使用叠氮核苷酸标记技术和CFTR膜片钳技术，将检验这一假设，即AMP和ATP与CFTR协同作用，AMP阻止ATP水解并诱导ATP：AMP磷酸化转移。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；它还有望确定AMP与CFTR结合的关键残基。该项目将为兰达克博士提供正式的职业指导和指导。迈克尔·J·威尔士博士将担任导师，杰弗里·C·默里博士、保罗·B·麦克雷博士和凯文·P·坎贝尔博士将担任顾问。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资金做好准备。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(见说明)：来自这些研究的知识将提供一个框架，以开发CFTR通道功能的激活剂和抑制剂。这些化合物可用于治疗与CFTR氯电流过少(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。(摘要结束)