Peptide Inhibitors Probe Structure and Function in Chloride Channels

摘要 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。的基因 编码这些通道的是与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， 靶向它们的药物制剂依赖于特异性的、高亲和力的 probes.本提案的目的是表征一种新的肽抑制剂， 与CFTR氯离子通道以高亲和力相互作用。CFTR存在缺陷， 囊性纤维化（CF）是一种致命的遗传性疾病，也在 多囊肾病（PKD）和分泌性腹泻。动物源肽毒素 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，还没有发现肽毒素与阴离子通道相互作用， 已知的分子身份。这个实验室最近分离出一种肽毒素， CFTR。新型毒素“GaTx 1”以状态依赖的方式抑制CFTR， 通道进入长期封闭状态。因此，GaTx 1代表了一个量子进步， 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的， GaTx 1的特性，在三个目标如下。目的1是鉴定野生型毒素 通过：使用单通道膜片钳和macropatch测定抑制动力学 通过纯化的CFTR细胞溶质记录、测定对ATP结合和水解的影响 结构域多肽，并询问GaTx 1是否抑制构象变化 通道孔本身的潜在门控。目的二是定位毒素的结合位点 通过一系列独立的研究， 随后进行定点诱变的方法。目标3是确定 通过使毒素本身突变，导致相互作用表面的鉴定。 该方法利用了一系列具有专业知识的高素质合作者 和私家侦探实验室的实验是互补的这项工作将提供独特的机会， 使用GaTx 1毒素作为研究工具，也可能有助于设计新的 用于CF、PKD、分泌性腹泻和涉及CFTR的其它病理的治疗剂。

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.