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.

抽象的氯化物通道在细胞生理的许多方面都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，并发展针对它们的药物剂依赖于特定的高亲和力的可用性探针。该建议的目的是表征一种新型的肽抑制剂与CFTR氯化物通道的高亲和力相互作用。 CFTR在致命的遗传疾病，囊性纤维化（CF），并且在多囊肾脏疾病（PKD）和分泌性腹泻。动物的肽毒素毒液是用于研究离子通道的最选择性和有用的工具之一。但是，到目前为止，还没有发现肽毒素与阴离子通道相互作用已知的分子身份。该实验室最近分离了一种抑制的肽毒素 CFTR。小说毒素“ GATX1”通过锁定以状态依赖的方式抑制CFTR 通道进入长期封闭状态。因此，GATX1代表了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。本应用程序提出了一系列目标在三个目标中表征GATX1，如下所示。目标1是表征野生型毒素作者：使用单通道贴片夹和Macropatch确定抑制作用的动力学记录，确定对ATP结合的影响和通过纯化的CFTR胞质的影响域多肽，询问GATX1是否抑制构象变化通道孔本身的基础门控。 AIM 2是定位毒素的绑定站点通过使用电生理和生化的一系列独立研究接下来是定向诱变的方法。目标3是确定的决定因素通过突变毒素本身的活动，从而鉴定相互作用的表面。该方法利用具有专业知识的高素质合作者列表与PI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颖 CF，PKD，分泌性腹泻和其他涉及CFTR的病理的治疗剂。