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.
| Mindell, Joseph A; Grigorieff, Nikolaus (2003) Accurate determination of local defocus and specimen tilt in electron microscopy. J Struct Biol 142:334-47 |
| Pirruccello, Michelle M; Grigorieff, Nikolaus; Mindell, Joseph A (2002) Electron diffraction of a bacterial ClC-type chloride channel. Novartis Found Symp 245:193-203; discussion 203-6, 261- |
