Mechanisms of CIC Chloride Transport Proteins

CIC 氯离子转运蛋白的机制

基本信息

批准号：
7267588
负责人：
Merritt C Maduke
金额：
$ 27.25万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-08-05 至 2010-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7267588
关键词：
Affect Biological Assay CIC inhibitor Cardiovascular system Carrier Proteins Charge Chloride Channels Chloride Ion Chlorides Condition Coupled Crystallography Cysteine Dependence Development Employee Strikes Engineering Epithelial Escherichia coli Family Family member Fluorescence Functional disorder Goals Helix (Snails)Homologous Gene Human Ions Light Lysine Maps Measures Mediating Membrane Methods Modeling Modification Movement Mutagenesis Mutation Neurons Pathway interactions Personal Satisfaction Physiology Population Proteins Protons Range Rate Reagent Research Personnel Resolution Scanning Side Site-Directed Mutagenesis Structure Sulfhydryl Compounds Testing Therapeutic Agents Vesicle Vestibule Work antiport antiporter base design disulfide bond ear helix electric field high throughput screening inhibitor/antagonist insight member mutant novel programs research study scaffold voltage

项目摘要

DESCRIPTION (provided by applicant): Members of the CIC family orchestrate the movements of chloride necessary for proper neuronal, muscular, cardiovascular, and epithelial function. Although certain eukaryotic CIC channels have been well characterized functionally, we remain in the dark about their detailed structures. Conversely, the structures of two prokaryotic CIC homologs were determined by x-ray crystallography, but we know little about their function. The high sequence identity between core invariant segments of prokaryotic and eukaryotic CICs argues for a shared structural scaffold, but the low (15-20%) overall identity also indicates that these families; differ in significant details. A striking functional correlate of the structural differences is the recent finding that CIC-ec1, an E. coli CIC, is not a chloride channel but a chloride-proton antiporter. Thus, the CIC family straddles an evolutionary cusp between these two different transport mechanisms. Our long-range goal is to determine how proton and chloride movement are coupled in CIC-ec1 and how this relates to proton- and chloride-activation of voltage-dependent gating in CIC-0. This work will shed light on the general mechanism of chloride permeation in the CIC family members, and will be essential for understanding the diverse physiology and pathophysiology seen amongst the CIC family members. In Specific Aim 1, we will test models of chloride-proton antiport using bilayer recordings and flux assays together with site-directed mutagenesis. Important technical developments include methods for obtaining oriented transporters, and the use of a high-throughput fluorescence-based transport assay to screen for specific inhibitors. In Specific aim 2, we will map the intracellular vestibule of CIC-0 using cysteine scanning mutagenesis. We will probe gating conformational changes by examining rates of cysteine modification under different conditions. Finally, we will analyze voltage- and proton-dependent gating at the single-channel level for mutations that cause significant changes in gating. Together, these studies will offer new insights into the functional relationships between channels and transporters.

描述（由申请人提供）：CIC家族的成员协调了适当的神经元，肌肉，心血管和上皮功能所需的氯化物运动。尽管某些真核CIC通道在功能上的表征很好，但我们仍然对其详细结构保持黑暗。相反，通过X射线晶体学确定了两个原核CIC同源物的结构，但我们对它们的功能知之甚少。原核生物和真核CIC的核心不变段之间的高序列身份主张共享结构性支架，但低（15-20％）的总体身份也表明这些家庭；重要细节有所不同。结构差异的引人注目的功能相关是最近的发现，即CIC-EC1（大肠杆菌CIC）不是氯化物通道，而是氯化物 - 普罗替型抗胞鼠。因此，CIC家族跨越了这两种不同的运输机制之间的进化尖。我们的远程目标是确定质子和氯化物运动如何在CIC-EC1中耦合，以及这与CIC-0中电压依赖性门控的质子和氯化物激活之间的关系。这项工作将阐明CIC家族成员中氯化物渗透的一般机制，对于理解CIC家族成员中看到的多种生理学和病理生理学至关重要。在特定的目标1中，我们将使用双层记录和通量测定以及定向诱变，测试氯化物 - 普罗替型对的模型。重要的技术发展包括用于获取定向转运蛋白的方法，以及使用高通量荧光的转运测定法对特定抑制剂进行筛选。在特定的目标2中，我们将使用半胱氨酸扫描诱变绘制CIC-0的细胞内前庭。我们将通过在不同条件下检查半胱氨酸修饰的速率来探测门控构象变化。最后，我们将分析单通道水平上的电压和质子依赖性门，以引起门控变化的突变。这些研究将共同提供有关渠道和运输者之间功能关系的新见解。