Peptide Inhibitors Probe Structure and Function in Chloride Channels

描述(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，以及开发针对它们的药物制剂，依赖于特定的、高亲和力的探针的可用性。这项建议的目标是表征一种与CFTR氯通道高度亲和力相互作用的新型多肽抑制剂。CFTR在致死性遗传性疾病囊性纤维化(CF)中存在缺陷，在多囊肾病(PKD)和分泌性腹泻中也起重要作用。动物毒素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，还没有发现与已知分子特性的阴离子通道相互作用的多肽毒素。该实验室最近分离出一种抑制CFTR的多肽毒素。这种新的毒素“GaTx1”通过将通道锁定到长时间关闭的状态，以状态依赖的方式抑制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GaTx1，其三个目标如下。目的1是通过单通道膜片钳和宏膜片记录来确定野生型毒素的特性：用单通道膜片钳和宏膜片记录来确定抑制动力学，用纯化的CFTR胞浆结构域多肽来测定对ATP结合和水解的影响，以及询问GaTx1是否抑制通道孔本身的门控的构象变化。目标2是通过一系列独立的研究来定位毒素的结合部位，方法是使用电生理和生化方法，然后进行定点突变。目标3是通过突变毒素本身来识别活性决定因素，从而识别相互作用的表面。这种方法利用了一批高度合格的合作者，他们的专业知识与PI的实验室相辅相成。这项工作将为将GaTx1毒素用作研究工具提供独特的机会，并可能有助于设计治疗CF、PKD、分泌性腹泻和其他涉及CFTR的病理的新疗法。与公共卫生相关：致命性遗传病囊性纤维化中的缺陷基因编码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。由于缺乏药理上有用的工具，这种与临床相关的蛋白质的结构和功能的研究一直受到阻碍。本实验室分离到一种CFTR的多肽毒素抑制剂，它以高亲和力和高特异性关闭通道；本提案旨在鉴定这种毒素GaTx1，并利用它来了解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.