C1C CHANNELS IN A HOMOGENEOUS EPITHELIUM

这个项目旨在解决有关ClC-通道家族的基本生物物理性质以及它们在分泌盐分的上皮细胞中所起的作用的问题。CLC通道在所有组织中都有表达，并已知在离子稳态中发挥重要作用，因为它们在人类盐分运输的遗传性疾病中起着重要作用，如巴特综合征和登特氏病。尽管它们很重要，但我们仍然对《中图法》家族的最基本特征一无所知。在这个提议中，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分泌盐分上皮的例子，但之前还没有人在这个组织中寻找ClC型通道基因。我的假设是ClC型氯离子通道有助于SRG分泌氯化钠，这是研究这些通道在分泌盐分上皮细胞中作用的理想系统。在前期工作中，L已经从SRG中克隆了四个ClC型氯离子通道的全长编码区。在这里，我建议详细研究其中的两个通道：Shark 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细胞中制备膜泡，并将它们融合到脂质双层膜中。类似的方法在这个实验室中对其他几个离子通道也是成功的。具体目标1.我建议确定SCLC-6和SCLC-7的单通道属性。这些实验将解决关于CLC家族分子结构的几个悬而未决的争议。特别是，他们将解决目前的争议，即一些CLC是否只有一条传导途径(与CLC-0相反，它有两条传导途径)。这些研究是对CLC家族进行详细结构-功能分析的前奏。在具体目标2中，我介绍了旨在确定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，这些通道可能位于SRG的顶膜；我将使用免疫化学染色来验证这一理论。无论这些通道的分布如何，这些通道的亚细胞定位将有助于深入了解它们的生理功能。我还将测试体内和体外直肠腺刺激对这种定位的影响。最后，在特定的目标3中，我将进行免疫共沉淀实验，以确定通道是否在体内和体外形成异构体复合体。了解功能通道的亚基组成是结构分析中的关键步骤。这些研究将极大地提高我们对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.