Mechanisms of CIC-Type Chloride Channels Function

CIC型氯离子通道功能机制

• 批准号: 6687147
• 负责人: JAMES E MELVIN
• 金额: $ 3.92万
• 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/6687147
• 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） 各种遗传性人类疾病和基因敲除小鼠模型都表明，电压激活的 CIC 氯 (CI-) 通道对于生存至关重要。 CIC基因家族不同成员的基因突变导致巴特综合征中的跨上皮离子转运受损，先天性肌强直中的肌肉兴奋性增加，登特病中的内体酸化和内吞作用减少，或骨石症中破骨细胞的细胞外酸化受损。此外，有针对性地破坏小鼠体内的几个 CIC 通道基因会导致失明。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 通道蛋白进行结构功能分析所需的结构框架，以了解其生物物理特性，包括这些通道被激活的机制。该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。我们将确定快门控过程和慢门控过程是否控制 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胞内和胞外[CI-]和[H+]的巨大变化如何影响门控。我们假设这些变化作为调节通道活动的反馈信号，如下所示：氯离子作为电荷提供者，调节门控的电压灵敏度；质子通过直接与 CIC-2 通道的门控机制相互作用来调节通道活动。因此，我们建议 H+ 结合位点的识别将定义 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的氨基酸。目标 2 和 3 旨在定义 CI- 和 H+ 对 CIC-2 CI- 通道门控以及赋予电压和 pH 敏感性的分子域的影响，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结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。这项研究将主要在墨西哥圣路易斯波托西自治大学与 Jorge Arreola 博士合作进行，作为 NIH 拨款 # R01 DE09692 的延伸。