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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.

摘要氯通道在细胞生理学的许多方面起着至关重要的作用。基因编码这些通道是与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，以及针对他们的药物制剂，依赖于特定的、高亲和力的可获得性探测器。这项提议的目标是表征一种新型的肽抑制剂，它可以与CFTR氯通道有高亲和力。CFTR存在缺陷，致命性遗传病，囊性纤维化(CF)，也在多囊肾病(PKD)和分泌性腹泻。动物来源的多肽毒素毒液是研究离子通道的最有选择性和最有用的工具之一；然而，到目前为止，还没有发现与阴离子通道相互作用的多肽毒素。已知的分子同一性。这个实验室最近分离出了一种能抑制 CFTR。新型毒素“GaTx1”通过锁定以一种状态依赖的方式抑制cftr 通道进入长时间关闭状态。因此，GaTx1代表了量子进步与结构探针相比，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以将GaTx1描述为以下三个目标。目标1是确定野生型毒素的特征应用单通道膜片钳和宏膜片技术测定抑制动力学记录、测定纯化的CFTR胞浆对三磷酸腺苷结合和水解的影响结构域多肽，并询问GaTx1是否抑制构象变化通道孔本身的下层栅极。目标2是定位毒素的结合部位通过一系列使用电生理和生化的独立研究随后是定点突变的方法。目标3是确定决定因素通过突变毒素本身来发挥活性，从而识别相互作用的表面。该方法利用了一批具有专业知识的高资质合作者是对私家侦探实验室的补充。这项工作将提供独特的机会使用GaTx1毒素作为研究工具，也可能有助于设计新的治疗CF、PKD、分泌性腹泻和其他涉及CFTR的病理。