peptide inhibitor: new probe for chloride channels

描述(申请人提供)：真核离子通道在细胞生理学的许多方面起着至关重要的作用。了解这些蛋白质的结构和功能依赖于特定的、高亲和力的探针的可用性。这个实验室的长期目标是确定两个结构无关、临床相关的阴离子通道的渗透和门控机制：CFTR通道和CIC通道家族。CFTR是囊性纤维化的主要缺陷所在，囊性纤维化是高加索人中最常见的致命性常染色体隐性遗传病，影响大约30,000美国人；CFTR在分泌性腹泻和多囊肾病(PKD)的病理生理中也是至关重要的。C1C通道几乎无处不在，在肾单位的几个节段中发挥着重要作用。尽管进行了大量研究，包括最近展示了两种原核生物C1C蛋白的晶体结构，但涉及渗透(CFTR和C1Cs)和门控(CICs)的结构尚不清楚，部分原因是缺乏适当的工具。多肽毒素已被证明是研究阳离子通透通道的最有选择性和最有用的工具之一。然而，没有这种多肽毒素被证明与已知分子特性的阴离子通道相互作用。本实验室的初步数据表明，蝎毒中含有一种或多种多肽成分，可抑制(S)cftr和C1c-2通道，尽管作用机制不同。这项R21提案寻求采取初步步骤来发展这些令人兴奋的观察结果，通过识别一个或多个活性毒素并使用它/它们来研究这些离子通道中门控的结构和机制。具体目标#1是收集关于蛇毒抑制作用的初步信息，然后利用这些信息形成生物检测方法，以指导活性成分的色谱分离和鉴定(S)。然后，特殊目的2号将使用分离的毒素(S)作为高亲和力探针来细致地研究CFTR和CIC-2通道。该实验计划结合了本实验室在使用结构/功能方法(通过电生理方法)研究CI通道方面的重要专业知识，以及合作者在使用最先进的色谱和光谱方法识别生物活性大分子方面的专业知识。拟议的探索性工作将允许以迄今为止不可能的方式研究这些重要的通道蛋白，并可能有助于设计治疗CF、分泌性腹泻、多囊肾病和其他肾病的新疗法。

DESCRIPTION (provided by applicant): Eukaryotic ion channels play crucial roles in many aspects of cell physiology. Understanding the structure and function of these proteins relies upon the availability of specific, high-affinity probes. The long-term goals of this laboratory are to determine the mechanisms of permeation and gating in two structurally unrelated, clinically relevant anion channels: the CFTR channel and the CIC family of channels. CFTR is the locus of the primary defect in cystic fibrosis (CF), the most common lethal, autosomal recessive disease among Caucasians, affecting approximately 30,000 Americans; CFTR is also crucial to the pathophysiologies of secretory diarrhea and polycystic kidney disease (PKD). C1C channels are nearly ubiquitous, and play important roles in several segments of the nephron. Despite significant study, including the recent demonstration of the crystal structure of two prokaryotic C1C proteins, the structures involved in permeation (for CFTR and C1Cs) and gating (for CICs) are not clear, partly due to lack of appropriate tools. Peptide toxins have proven to be among the most selective and useful tools for the study of cation-permeable channels. However, no such peptide toxins have been shown to interact with anion channels of known molecular identity. Preliminary data from this laboratory has now shown that scorpion venom contains a peptide component, or components, that inhibit(s) CFTR and C1C-2channels, albeit with different mechanism. This R21 proposal seeks to take the initial steps to develop these exciting observations, by identifying the active toxin or toxins and using it/them to study the architecture and mechanisms of gating in these ion channels. Specific Aim #1 is to gather initial information on inhibition by venom, then use this to form bioassays to guide the chromatographic separation and identification of the active component(s). Specific Aim #2 will then use the isolated toxin(s) as a high-affinity probe to study CFTR and CIC-2 channels in exquisite detail. The experimental plan combines the significant expertise of this laboratory in using structure/function approaches (by electrophysiological methods) to study CI channels with the expertise of collaborators in using state-of-the-art chromatographic and spectrometric approaches for the identification of bioactive macromolecules. The proposed exploratory work will allow the study of these important channel proteins in ways heretofore impossible, and will likely aid in the design of novel therapies for CF, secretory diarrhea, polycystic kidney disease, and other nephropathies.