Opening and Closing Mechanisms of CFTR Channels

CFTR通道的开启和关闭机制

基本信息

批准号：
7236335
负责人：
Laszlo Csanady
金额：
$ 3.25万
依托单位：
SEMMELWEIS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-05-15 至 2010-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7236335
关键词：
ABCC1 gene ATP Hydrolysis ATP-Binding Cassette Transporters Address Adenylyl Imidodiphosphate Algorithms Antigen Presentation Apical Bacteria Binding Biomedical Research Catalytic Domain Chemotherapy-Oncologic Procedure Chloride Channels Cholesterol Collaborations Complex Cyclic AMP-Dependent Protein Kinases Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Defect Dependence Discrimination Disease Disruption Dissection Drug Metabolic Detoxication Drug Transport Epithelium Event Excision Exposure to Family Family member Genes Goals Grant Human Hungary Hydrolysis Individual Inherited Ion Channel Ionic Strengths Ions Kinetics Learning Link Modeling Molecular Molecular Conformation Multi-Drug Resistance Mutate Mutation Nucleotides Organism P-Glycoprotein P-Glycoproteins Pattern Phosphorylation Physiological Processes Production Protein Kinase Protein Overexpression Proteins Range Rate Relative (related person)Research Resolution Scheme Serine Site Sodium Chloride Solutions Structure Surface System Temperature Time Transmembrane Domain United States National Institutes of Health Universities Walkers Water Movements Xenopus oocyte analog analytical tool base bile salts cell type computer program cystic fibrosis patients dimer enthalpy genetic regulatory protein human disease insulin secretion man mutant neoplastic cell novel parent grant patch clamp receptor single molecule

项目摘要

DESCRIPTION (provided by applicant): This application is to continue support for productive collaborative research done largely by the foreign PI, Laszlo Csanady, at Semmelweis University in Budapest, Hungary, to extend NIH grant R01 DK051767 of the US collaborator, David C. Gadsby. The parent grant aims to understand molecular mechanisms that control opening and closing CFTR (cystic fibrosis transmembrane conductance regulator) Cl- ion channels, encoded by the gene mutated in CF patients. CFTR is one of -50 human ABC (ATP-binding cassette) proteins, many (like SUR, the surfonylurea receptor, or P-glycoprotein and MRP, the multidrug resistance related protein) involved in human disease. CFTR is the sole ABC protein to form an ion channel, and so is the only one amenable to high-resolution studies of single-molecule function. But, statistical comparison of >15,000 available ABC sequences argues that certain functional mechanisms elucidated in CFTR will have broad applicability in other ABC proteins: this includes how ATP-driven events in the nucleotide-binding domains (NBDs) control conformational changes in the transmembrane domains that in CFTR open and close the ion pore. We have proposed that once ATP has bound to both NBDs of a closed CFTR channel the NBDs dimerize, burying the ATPs in the dimer interface, prompting channel opening: hydrolysis of one of the ATPs leads to disruption of the NBD dimer, and to channel closing. To further clarify this gating cycle, the goal of the research proposed here is to use single-channel kinetic analysis to identify all conformational states of a CFTR channel and the connectivities among them. We will use a novel statistical analysis we recently developed to dissect the two consecutive steps expected to occur in every open burst of a CFTR channel. To learn the molecular structural underpinnings of those steps, we will apply our analysis to wild-type and to select mutant CFTR channels while varying temperature, ionic strength, degree of regulatory PKA phosphorylation, and structure of activating nucleotide (i.e. ATP and/or analogs). We will also evaluate the newly uncovered complex gating of a non-hydrolytic mutant CFTR (D1370N). Finally, we will determine the molecular basis for the locked-open conformation of CFTR channels observed after exposing them to a mixture of ATP plus non-hydrolyzable ATP analog (e.g. AMPPNP). We will record from inside-out patches, excised from Xenopus oocytes expressing CFTR, in a fast-flow system with accurate temperature control.

描述（由申请人提供）：本申请旨在继续支持主要由外国 PI Laszlo Csanady 在匈牙利布达佩斯 Semmelweis 大学完成的富有成效的合作研究，以延长美国合作者 David C. Gadsby 的 NIH 拨款 R01 DK051767 。母基金旨在了解控制 CFTR（囊性纤维化跨膜电导调节器）Cl-离子通道打开和关闭的分子机制，该通道由 CF 患者突变的基因编码。 CFTR 是 -50 人类 ABC（ATP 结合盒）蛋白之一，许多蛋白（如 SUR（磺脲类受体）或 P-糖蛋白和 MRP（多药耐药性相关蛋白））与人类疾病有关。 CFTR 是唯一形成离子通道的 ABC 蛋白，因此也是唯一适合单分子功能高分辨率研究的蛋白。但是，对超过 15,000 个可用 ABC 序列的统计比较表明，CFTR 中阐明的某些功能机制将在其他 ABC 蛋白中具有广泛的适用性：这包括核苷酸结合域 (NBD) 中 ATP 驱动的事件如何控制跨膜域的构象变化CFTR 中离子孔的打开和关闭。我们提出，一旦 ATP 与封闭 CFTR 通道的两个 NBD 结合，NBD 就会二聚，将 ATP 埋入二聚体界面中，从而促进通道打开：其中一个 ATP 的水解导致 NBD 二聚体破坏，并导致通道关闭。为了进一步阐明这个门控循环，本文提出的研究目标是使用单通道动力学分析来识别CFTR通道的所有构象状态以及它们之间的连接性。我们将使用最近开发的一种新颖的统计分析来剖析 CFTR 通道的每个开放突发中预期发生的两个连续步骤。为了了解这些步骤的分子结构基础，我们将分析应用于野生型并选择突变型 CFTR 通道，同时改变温度、离子强度、调节 PKA 磷酸化程度和激活核苷酸（即 ATP 和/或类似物）的结构）。我们还将评估新发现的非水解突变体 CFTR (D1370N) 的复杂门控。最后，我们将确定 CFTR 通道暴露于 ATP 加不可水解 ATP 类似物（例如 AMPPNP）的混合物后观察到的锁定开放构象的分子基础。我们将在具有精确温度控制的快速流动系统中从内到外记录从表达 CFTR 的非洲爪蟾卵母细胞中切下的斑块。