C1C CHANNELS IN A HOMOGENEOUS EPITHELIUM

均质上皮中的 C1C 通道

• 批准号: 6516762
• 负责人: Joseph A Mindell
• 金额: $ 4.67万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6516762
• 关键词: biochemistry; chloride channels; electrolyte balance; electrophysiology; epithelium; hormone regulation /control mechanism; immunocytochemistry; immunoprecipitation; protein localization; protein structure function; secretion; sharks; sodium chloride; tissue /cell culture; transfection; voltage /patch clamp

This project is designed to attack questions regarding the basic biophysical properties of the ClC family of Cl- channels and the roles they play in salt-secreting epithelia. ClC channels are represented in all tissues, and are known to play a prominent part in ionic homeostasis by virtue of their contributions to human genetic disorders of salt transport, such as Bartter's syndrome and Dent's disease. Despite their importance, we remain remarkably ignorant of even the most basic features of the ClC family. In this proposal, I present a powerful new system, the shark rectal gland (SRG), to probe the architecture of ClC-type channels and their roles in electrolyte physiology. The SRG is perhaps the best-studied example of a salt-secreting epithelium, yet no one has previously looked for ClC-type channel genes in this tissue. My hypothesis is that ClC-type chloride channels contribute to NaCl secretion in SRG, and that this is an ideal system to study the role of these channels in salt-secreting epithelia. In preliminary work, l have cloned from the SRG four full length cDNA's encodinghomologs of ClC-type Cl channels. Here I propose to study two of these channels in detail: shark ClC-6 and ClC-7. I will take a new approach to functional expression (for these channels), preparing membrane vesicles from HEK 293 cells expressing sClC-6 and sClC-7 and fusing them into lipid bilayer membranes. Similar methodology has been successful in this lab for several other ion channels. Specific Aim 1. I propose to determine the single-channel properties of sClC-6 and sClC-7. These experiments will resolve several outstanding controversies regarding the molecular architecture of the ClC family. In particular, they will resolve the current dispute as to whether some ClC's have only one conduction pathway (in contrast to ClC-0, which has two). Such studies are a prelude to detail structure-function analysis of the ClC family. In Specific Aim 2, I present experiments designed to determine the subcellular localization o f sClC-6 and sClC-7 in their native tissue. Current data suggest that these channels may be in the apical membrane of SRG; I will test this theory using immunochemical staining. Whatever the distribution, the subcellular localization of these channels will yield insight into their physiological function. I will also test the effects on this localization of in vivo and in vitro rectal gland stimuli. Finally, in Specific Aim 3, I will perform coimmunoprecipiation experiments to determine whether the channels form heteromeric complexes, both in vivo and in vitro. Understanding the subunit composition of the functional channel is a critical step in structural analysis. These studies will dramatically improve our understanding of the role played by ClC channels in normal salt-secretion and will establish a paradigm for the eventual development of drugs to modulate these processes.

这个项目的目的是攻击有关的Cl-通道的ClC家族的基本生物物理特性和盐分泌上皮细胞中发挥的作用的问题。 C1 C通道存在于所有组织中，并且已知由于其对盐转运的人类遗传病症（例如巴特综合征和登特病）的贡献而在离子稳态中起重要作用。 尽管它们很重要，但我们仍然对CLC家族的最基本特征一无所知。 在这个建议中，我提出了一个强大的新系统，鲨鱼直肠腺（SRG），探索ClC型通道的结构和它们在电解质生理学中的作用。SRG可能是盐分泌上皮细胞中研究得最好的例子，但以前没有人在这种组织中寻找ClC型通道基因。我的假设是，ClC型氯离子通道有助于SRG中的NaCl分泌，并且这是研究这些通道在盐分泌上皮中的作用的理想系统。 在前期工作中，我从SRG中克隆了四个ClC型Cl通道的全长cDNA编码同源物。 在这里，我建议详细研究其中的两个通道：鲨鱼ClC-6和ClC-7。 我将采取一种新的方法来功能表达（对于这些通道），从表达sClC-6和sClC-7的HEK 293细胞中制备膜囊泡，并将它们融合到脂质双层膜中。 类似的方法在本实验室中已成功用于其他几种离子通道。 具体目标1。 我建议确定sClC-6和sClC-7的单通道特性。这些实验将解决几个突出的争议，关于CLC家族的分子结构。 特别是，他们将解决目前的争议，一些CLC是否只有一个传导途径（与CLC-0相比，有两个）。 这些研究是详细的CLC家族的结构-功能分析的前奏。 在具体目标2中，我提出了旨在确定sClC-6和sClC-7在其天然组织中的亚细胞定位的实验。 目前的数据表明，这些通道可能是在SRG的顶膜，我将测试这一理论使用免疫化学染色。 无论分布如何，这些通道的亚细胞定位将使我们深入了解它们的生理功能。 我还将测试在体内和体外直肠腺刺激对这种定位的影响。 最后，在具体目标3，我将进行coimmunoptimization实验，以确定是否通道形成异聚体复合物，在体内和体外。 了解功能通道的亚基组成是结构分析的关键步骤。 这些研究将大大提高我们对CLC通道在正常盐分泌中所起作用的理解，并将为最终开发调节这些过程的药物建立一个范例。

项目成果

Electron diffraction of a bacterial ClC-type chloride channel.

细菌 ClC 型氯离子通道的电子衍射。

• 发表时间: 2002
• 期刊: Novartis Foundation symposium.
• 作者: Pirruccello,MichelleM;Grigorieff,Nikolaus;Mindell,JosephA
• 通讯作者: Mindell,JosephA