Peptide Inhibitors Probe Structure and Function in Chloride Channels

肽抑制剂探测氯离子通道的结构和功能

基本信息

批准号：
8061578
负责人：
NAEL A MCCARTY
金额：
$ 30.96万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-01 至 2014-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8061578
关键词：
ATP Hydrolysis Affinity American Amino Acids Animals Anions Architecture Binding Binding Sites Biochemical Biological Biological Assay Cations Cell physiology Characteristics Charge Chemistry Chloride Channels Chlorotoxin Collaborations Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Development Diarrhea Differential Scanning Calorimetry Disease Disulfides Electrophysiology (science)Elements Epithelial Cells Future Genes Glutathione Goals Hereditary Disease Human Hydrolysis Individual Ion Channel Kinetics Laboratories Lead Mediating Membrane Proteins Modeling Molecular Molecular Conformation Molecular Models Mutate Mutation Names National Institute of Diabetes and Digestive and Kidney Diseases Pathology Peptides Pharmacologic Substance Phase Photoaffinity Labels Play Polycystic Kidney Diseases Proteins Qualifying Reporting Research Role Series Site Site-Directed Mutagenesis Solid Specificity Structural Biologist Structure Surface Techniques Time Toxin Ursidae Family Venoms Work base cell type clinically relevant cystic fibrosis patients design experience human disease inhibitor/antagonist insight molecular modeling mutant novel novel therapeutics patch clamp peptidomimetics polypeptide protein structure function public health relevance quantum research study therapeutic target tool

项目摘要

DESCRIPTION (provided by applicant): Chloride channels play crucial roles in many aspects of cell physiology. The genes encoding these channels are the loci for several human diseases relevant to the NIDDK. Understanding the structure and function of these proteins, and development of pharmaceutical agents targeting them, relies upon the availability of specific, high-affinity probes. The goal of this proposal is to characterize a novel peptide inhibitor which interacts with high affinity with the CFTR chloride channel. CFTR is defective in the lethal genetic disease, Cystic Fibrosis (CF), and also plays an important role in polycystic kidney disease (PKD) and secretory diarrhea. Peptide toxins from animal venom are among the most selective and useful tools for the study of ion channels; however, until now, no peptide toxins have been found that interact with anion channels of known molecular identity. This laboratory recently isolated a peptide toxin that inhibits CFTR. The novel toxin, "GaTx1", inhibits CFTR in a state-dependent manner by locking channels into a long closed state. Hence, GaTx1 represents a quantum advance in how we can approach structure/function studies in CFTR, compared to the structural probes currently available. The present application proposes a series of objectives to characterize GaTx1, in three aims as follows. Aim 1 is to characterize the wildtype toxin by: determining kinetics of inhibition using single-channel patch clamp and macropatch recording, determining effects on ATP binding and hydrolysis by purified CFTR cytosolic domain polypeptides, and asking whether GaTx1 inhibits the conformational change underlying gating of the channel pore itself. Aim 2 is to localize the toxin's binding site by a series of independent studies using electrophysiological and biochemical approaches followed by site-directed mutagenesis. Aim 3 is to identify determinants of activity by mutating the toxin itself, leading to identification of the interacting surfaces. The approach takes advantage of a list of highly qualified collaborators with expertise complementary to that of the PI's lab. This work will provide the unique opportunity to use the GaTx1 toxin as a research tool, and also will likely aid in the design of novel therapeutics for CF, PKD, secretory diarrhea, and other pathologies that involve CFTR. PUBLIC HEALTH RELEVANCE: The gene defective in the lethal genetic disease cystic fibrosis encodes the CFTR protein, which forms a chloride ion channel expressed in many epithelial cell types. Studies of the structure and function of this clinically-relevant protein have been hampered by the lack of pharmacologically useful tools. This laboratory has isolated a peptide toxin inhibitor of CFTR, which locks the channel closed with high affinity and high specificity; the present proposal aims to characterize the toxin, GaTx1, and to use it to understand how the CFTR channel protein is regulated.

描述（由申请人提供）：氯离子通道在细胞生理学的许多方面发挥着至关重要的作用。编码这些通道的基因是与 NIDDK 相关的几种人类疾病的基因座。了解这些蛋白质的结构和功能以及针对它们的药剂的开发依赖于特异性、高亲和力探针的可用性。该提案的目标是表征一种新型肽抑制剂，该抑制剂与 CFTR 氯离子通道具有高亲和力相互作用。 CFTR 在致命性遗传病囊性纤维化 (CF) 中存在缺陷，并且在多囊肾病 (PKD) 和分泌性腹泻中也发挥着重要作用。来自动物毒液的肽毒素是研究离子通道最具选择性和最有用的工具之一；然而，到目前为止，尚未发现肽毒素与已知分子特性的阴离子通道相互作用。该实验室最近分离出一种抑制CFTR的肽毒素。这种新型毒素“GaTx1”通过将通道锁定为长时间关闭状态，以状态依赖性方式抑制 CFTR。因此，与目前可用的结构探针相比，GaTx1 代表了我们在 CFTR 中进行结构/功能研究的量子进步。本申请提出了一系列目标来表征GaTx1，包括以下三个目标。目标 1 是通过以下方式表征野生型毒素：使用单通道膜片钳和大膜片记录确定抑制动力学，确定纯化的 CFTR 胞质结构域多肽对 ATP 结合和水解的影响，并询问 GaTx1 是否抑制门控下的构象变化。通道孔本身。目标 2 是通过一系列独立研究，使用电生理学和生化方法，然后进行定点诱变，定位毒素的结合位点。目标 3 是通过突变毒素本身来识别活性的决定因素，从而识别相互作用的表面。该方法利用了一系列高素质合作者，他们的专业知识与 PI 实验室的专业知识互补。这项工作将为使用 GaTx1 毒素作为研究工具提供独特的机会，并且也可能有助于设计针对 CF、PKD、分泌性腹泻和涉及 CFTR 的其他病理的新型疗法。公共健康相关性：致命性遗传病囊性纤维化的缺陷基因编码 CFTR 蛋白，该蛋白形成在许多上皮细胞类型中表达的氯离子通道。由于缺乏药理学上有用的工具，对这种临床相关蛋白质的结构和功能的研究受到了阻碍。本实验室分离出一种CFTR的肽毒素抑制剂，其锁定通道关闭，具有高亲和力和高特异性；目前的提案旨在表征毒素 GaTx1，并用它来了解 CFTR 通道蛋白是如何调节的。