Mechanisms of CIC-Type Chloride Channels Function

CIC型氯离子通道功能机制

• 批准号: 6941272
• 负责人: JAMES E MELVIN
• 金额: $ 3.2万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6941272
• 关键词: Mexico; acinar cell; aminoacid; binding sites; biological signal transduction; cell morphology; chemical kinetics; chloride channels; chloride ion; confocal scanning microscopy; electrophysiology; hydrogen ions; in situ hybridization; ion transport; membrane proteins; protein localization; protein structure function; saliva; secretion; site directed mutagenesis; sodium chloride; tissue /cell culture; transport proteins; voltage /patch clamp; voltage gated channel

DESCRIPTION (provided by applicant) Voltage-activated, CIC chloride (CI-) channels are essential for survival as illustrated by various inherited human diseases and knockout mouse models. Genetic mutations of distinct members of the CIC gene family lead to either impaired transepithelial ion transport in Bartter's syndrome, to increased muscle excitability in myotonia congenita, to reduced endosomal acidification and endocytosis in Dent's disease, or to impaired extracellular acidification by osteoclasts in osteopetrosis. Moreover, targeted disruption of several CIC channel genes in mice results in blindness. The three-dimensional structure of bacterial CIC channels determined by X-ray analysis provides the structural framework needed to perform structure-function analysis of CIC channel proteins in order to understand their biophysical properties, including the mechanism by which these channels are activated. This project will determine the gating and modulation of the CIC-2 CI-channel cloned from mouse parotid acinar cells when expressed in human kidney cells. We will determine if fast and slow gating processes control the kinetics of CIC-2, as shown for CIC-0 and CIC-1. Next, we will investigate how the large changes in intracellular and extracellular [CI-] and [H+] that epithelial cells undergo affect gating. We hypothesize that these changes serve as feedback signals that regulate channel activity as follows: chloride ions, by serving as a charge provider, regulate the voltage sensitivity of gating; and protons regulate channel activity by interacting directly with the gating machinery of the CIC-2 channel. Thus, we propose that the identification of the H+ binding sites will define the structural determinants of CIC-2 gating. The experiments proposed in Aim 1 will study the gating of CIC-2 and identify amino acids that form the gates of the channel. Aims 2 and 3 are designed to define the effects of CI- and H+ on CIC-2 CI- channel gating, as well as the molecular domains conferring voltage and pH sensitivity with the ultimate goal of defining the structure(s) responsible for gating. A combination of electrophysiological, molecular biology and amino acid-labeling techniques will be used to perform structure-function analysis of the channel protein and to track changes in gating, CI- and H+ sensitivity. This research will be carried out primarily at Universidad Autonoma de San Luis Potosi, Mexico in collaboration with Dr. Jorge Arreola as an extension of NIH grant # R01 DE09692.

描述（由申请人提供） 电压激活的CIC氯化物（CI-）通道对于生存至关重要，如各种遗传性人类疾病和敲除小鼠模型所示。 CIC基因家族的不同成员的遗传突变会导致Bartter综合征中的旋转离子传输受损，从而增加了肌张力的肌肉兴奋性，从而减少了Dent疾病的内体酸化和内吞作用，或者通过Osteocopetss sosteopopeastis in osteopopopopoopopopetsis insosteasis in osteoopopopopects in osteoopopeopopeopopects in osteoopopopopopects in osteopopects in osteopopects in osteoopopects in osteopects in osteopopect。此外，针对小鼠中几个CIC通道基因的靶向破坏会导致失明。通过X射线分析确定的细菌CIC通道的三维结构提供了对CIC通道蛋白进行结构函数分析所需的结构框架，以了解其生物物理特性，包括激活这些通道的机制。该项目将确定在人肾细胞中表达时从小鼠腮腺细胞克隆的CIC-2 CI渠道的门控和调节。我们将确定快速和缓慢的门控过程是否控制CIC-2的动力学，如CIC-0和CIC-1所示。接下来，我们将研究上皮细胞经历的细胞内和细胞外[CI-]和[H+]的大变化如何影响门控。我们假设这些变化用作反馈信号，如以下方式调节通道活动：氯离子通过充电提供商来调节门控的电压敏感性；质子和质子通过与CIC-2通道的门控机械直接相互作用来调节通道活性。因此，我们建议识别H+结合位点将定义CIC-2门控的结构决定因素。 AIM 1中提出的实验将研究CIC-2的门控，并鉴定构成通道门的氨基酸。 AIM 2和3旨在定义CI-2和H+对CIC-2 CI-通道门控的影响，以及赋予电压和pH值敏感性的分子结构域，以定义负责门控的结构的最终目标。电生理，分子生物学和氨基酸标记技术的结合将用于对通道蛋白的结构 - 函数分析，并跟踪门控，CII和H+灵敏度的变化。这项研究将主要在墨西哥的San Luis Potosi大学与Jorge Arreola博士合作，作为NIH授予＃R01 DE09692的延伸。