A peptide inhibitor: new probe for chloride channels

描述（由申请人提供）：真核细胞离子通道在细胞生理学的许多方面发挥关键作用。了解这些蛋白质的结构和功能依赖于特异性，高亲和力探针的可用性。本实验室的长期目标是确定两个结构无关的，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（CF）的原发性缺陷的位点，囊性纤维化是高加索人中最常见的致死性常染色体隐性疾病，影响约30，000名美国人; CFTR对分泌性腹泻和多囊肾病（PKD）的病理生理也至关重要。C1 C通道几乎是普遍存在的，并且在肾单位的多个节段中发挥重要作用。尽管进行了大量研究，包括最近证明了两种原核C1 C蛋白的晶体结构，但涉及渗透（CFTR和C1 C）和门控（CIC）的结构尚不清楚，部分原因是缺乏适当的工具。肽毒素已被证明是研究阳离子渗透通道的最具选择性和最有用的工具之一。然而，没有这样的肽毒素已被证明与已知分子身份的阴离子通道相互作用。该实验室的初步数据显示，蝎毒含有一种或多种肽成分，可以抑制CFTR和C1 C-2通道，尽管机制不同。这项R21提案旨在采取初步措施，通过识别活性毒素或毒素并使用它/它们来研究这些离子通道中的门控结构和机制，来开发这些令人兴奋的观察结果。具体目标#1是收集关于毒液抑制的初始信息，然后使用这些信息形成生物测定，以指导色谱分离和活性组分的鉴定。然后，特异性目标#2将使用分离的毒素作为高亲和力探针，以细致地研究CFTR和CIC-2通道。该实验计划结合了该实验室在使用结构/功能方法（通过电生理学方法）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鉴定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.