C1C CHANNELS IN A HOMOGENEOUS EPITHELIUM

本项目旨在研究ClC Cl-通道家族的基本生物物理特性及其在盐分泌上皮中的作用。ClC通道在所有组织中都存在，并且由于它们对人类盐运输遗传疾病（如Bartter综合征和Dent's病）的贡献，已知在离子稳态中起着重要作用。尽管它们很重要，但我们仍然对ClC家族最基本的特征一无所知。在本提案中，我提出了一个强大的新系统，鲨鱼直肠腺（SRG），以探索clc型通道的结构及其在电解质生理中的作用。SRG可能是研究得最好的盐分泌上皮的例子，但以前没有人在该组织中寻找clc型通道基因。我的假设是，在SRG中，clc型氯离子通道参与了盐的分泌，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4个全长cDNA的clc型Cl通道编码同源物。在这里，我建议详细研究其中的两个通道：鲨鱼的ClC-6和ClC-7。我将采用一种新的功能表达方法（针对这些通道），从表达sClC-6和sClC-7的HEK 293细胞中制备膜囊泡，并将其融合到脂质双分子层膜中。类似的方法在本实验室已经成功地用于其他几个离子通道。具体目标我建议确定sClC-6和sClC-7的单通道特性。这些实验将解决关于ClC家族分子结构的几个突出争议。特别是，它们将解决目前关于某些ClC是否只有一条传导途径的争议（与ClC-0相反，ClC-0有两条传导途径）。这些研究是ClC家族详细结构功能分析的前奏。在Specific Aim 2中，我提出了旨在确定sClC-6和sClC-7在其原生组织中的亚细胞定位的实验。目前的资料表明，这些通道可能位于SRG的顶膜中；我会用免疫化学染色来验证这个理论。无论分布如何，这些通道的亚细胞定位将有助于了解它们的生理功能。我还将测试体内和体外直肠刺激对这种定位的影响。最后，在Specific Aim 3中，我将进行共免疫沉淀实验，以确定这些通道是否在体内和体外形成异质复合物。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。

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.