Mechanisms of CIC-Type Chloride Channels Function

描述（由申请人提供） 电压激活的CIC氯化物（Cl-）通道对于存活是必不可少的，如各种遗传性人类疾病和敲除小鼠模型所示。CIC基因家族不同成员的基因突变导致Bartter综合征中跨上皮离子转运受损，先天性肌强直中肌肉兴奋性增加，Dent病中内体酸化和内吞作用减少，或骨硬化症中破骨细胞胞外酸化受损。此外，小鼠中几种CIC通道基因的靶向破坏导致失明。通过X射线分析确定的细菌CIC通道的三维结构提供了进行CIC通道蛋白的结构-功能分析所需的结构框架，以了解它们的生物物理特性，包括这些通道被激活的机制。本项目将确定从小鼠腮腺腺泡细胞克隆的CIC-2 CI通道在人肾细胞中表达时的门控和调节。我们将确定快速和慢速门控过程是否控制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+]的巨大变化如何影响门控。我们假设，这些变化作为反馈信号，调节通道活性如下：氯离子，作为一个电荷提供者，调节门控的电压敏感性;和质子调节通道活性直接与CIC-2通道的门控机械相互作用。因此，我们建议，H+结合位点的鉴定将定义CIC-2门控的结构决定因素。目标1中提出的实验将研究CIC-2的门控并鉴定形成通道门控的氨基酸。目的2和3旨在定义Cl-和H+对CIC-2 Cl-通道门控的影响，以及赋予电压和pH敏感性的分子结构域，最终目标是定义负责门控的结构。电生理学、分子生物学和氨基酸标记技术的组合将用于对通道蛋白进行结构-功能分析，并跟踪门控、CI-和H+敏感性的变化。本研究将主要在墨西哥圣刘易斯波托西自治大学与Jorge Arreola博士合作进行，作为NIH资助#R 01 DE 09692的延伸。

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.