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：
8460503
负责人：
NAEL A MCCARTY
金额：
$ 31.63万
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 2015-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8460503
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 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 quantum research study therapeutic target tool

项目摘要

ABSTRACT 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.

抽象的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。基因编码这些通道的是与 NIDDK 相关的几种人类疾病的位点。了解这些蛋白质的结构和功能，并开发针对它们的药剂依赖于特定的、高亲和力的可用性探针。该提案的目标是表征一种新型肽抑制剂，该抑制剂与 CFTR 氯通道具有高亲和力相互作用。 CFTR 有缺陷致命的遗传病，囊性纤维化（CF），也在多囊肾病（PKD）和分泌性腹泻。来自动物的肽毒素毒液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，尚未发现与阴离子通道相互作用的肽毒素已知分子身份。该实验室最近分离出一种肽毒素，可抑制 CFTR。新型毒素“GaTx1”通过锁定以状态依赖性方式抑制 CFTR 通道进入长时间关闭状态。因此，GaTx1 代表了在如何与结构探针相比，我们可以在 CFTR 中进行结构/功能研究目前可用。本申请提出了一系列目标描述 GaTx1 的特征，有以下三个目标。目标 1 是表征野生型毒素通过：使用单通道膜片钳和大膜片确定抑制动力学记录、确定纯化 CFTR 胞质对 ATP 结合和水解的影响域多肽，并询问 GaTx1 是否抑制构象变化通道孔本身的底层门控。目标 2 是定位毒素的结合位点通过一系列使用电生理学和生物化学的独立研究随后进行定点诱变的方法。目标 3 是确定通过突变毒素本身来识别活性，从而识别相互作用的表面。该方法利用了一系列具有专业知识的高素质合作者与 PI 实验室的补充。这项工作将为使用 GaTx1 毒素作为研究工具，也可能有助于设计新颖的 CF、PKD、分泌性腹泻和其他涉及 CFTR 的病理的治疗方法。