Peptide Inhibitors Probe Structure and Function in Chloride Channels

描述（由申请人提供）：氯离子通道在细胞生理学的许多方面起着至关重要的作用。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，以及靶向它们的药物制剂的开发，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与CFTR氯离子通道以高亲和力相互作用的新型肽抑制剂。CFTR在致死性遗传病囊性纤维化（CF）中有缺陷，并且在多囊肾病（PKD）和分泌性腹泻中也起重要作用。来自动物毒液的肽毒素是研究离子通道的最具选择性和最有用的工具之一;然而，到目前为止，还没有发现肽毒素与已知分子身份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。新型毒素“GaTx 1”通过将通道锁定在长时间关闭状态，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，GaTx 1代表了我们如何在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GaTx 1，三个目标如下。目的1是野生型毒素的特点：确定抑制动力学，使用单通道膜片钳和macropatch记录，确定对ATP的结合和水解纯化CFTR胞质结构域多肽的影响，并询问是否GaTx 1抑制潜在的门控通道孔本身的构象变化。目的2是定位毒素的结合位点，通过一系列独立的研究，使用电生理和生物化学的方法，然后定点突变。目的3是通过突变毒素本身来鉴定活性的决定因素，从而鉴定相互作用的表面。该方法利用了一系列高素质的合作者，他们的专业知识与PI实验室的专业知识互补。这项工作将提供使用GaTx 1毒素作为研究工具的独特机会，也可能有助于设计CF，PKD，分泌性腹泻和其他涉及CFTR的病理学的新疗法。公共卫生相关性：在致死性遗传疾病囊性纤维化中有缺陷的基因编码CFTR蛋白，其形成在许多上皮细胞类型中表达的氯离子通道。这种临床相关蛋白质的结构和功能的研究一直受到缺乏实用工具的阻碍。该实验室已经分离出CFTR的肽毒素抑制剂，其以高亲和力和高特异性锁定通道关闭;本提案旨在表征毒素GaTx 1，并使用它来了解CFTR通道蛋白是如何调节的。

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.