It is believed that the basis of cystic fibrosis (CF) is abnormal regulation of epithelial ion and fluid transport. Although all the functions of the CF gene product, CFTR, may not yet be established, recent evidence has demonstrated that CFTR is a cAMP-regulated Cl1- channel. Many mutations in CFTR have been described, but the most common is a deletion of phenylalanine at residue 508 (deltaF508-CFTR). Expression of deltaF508-CFTR does not confer plasma membrane Cl- permeability. Recent studies of the biosynthetic processing and cellular localization of CFTR suggest that deltaF508-CFTR fails to reach the plasma membrane. DeltaF508-CFTR is fully translated but lacks Golgi- mediated glycosylation and therefore appears to be retained in the endoscopic reticulum (ER). In some cells grown at low temperatures, expression of deltaF508-CFTR does result in plasma membrane Cl- channel activity. Because the mechanism underlying ER retention have not been determined, it is unknown whether they abolish or alter the Cl-channel activity of CFTR. Thus, the established Cl- channel functionality of deltaF508-CFTR raises the question of whether it functions in the ER. The consequences of deltaF508-CFTR Cl-channel activity or lack thereof in the ER are unknown because it has not been determined if wild-type CFTR functions there. Abnormal Cl- channel activity may affect the ER membrane potential and ionic composition. Since the ER is the site of synthesis and processing of proteins and lipids and is a Ca2+ reservoir in cells, such effects may have important consequences. An important issue is to determine whether CFTR and mutant CFTRs which are trapped in the ER, in particular deltaF508-CFTR, are active there. Although reconstitution techniques have been employed traditionally to analyze intracellular ion channels, a significant disadvantage is that the channels no longer exist within their normal milieu, with its unique protein and lipid composition. Thus, protein-protein interactions which might be important for CFTR function, stability or trafficking might be disrupted, and specific regulation and activity observed in bilayers may not faithfully mimic the situation in situ. The purpose of this research is to develop a novel system to examine CFTR channel function, regulation and expression in ER membranes, based on the Xenopus oocyte expression system and the fact that the outer membrane of the nuclear envelope is in continuity with the ER. By patch clamping isolated Xenopus oocyte nuclei, the oocyte expression system will be exploited to examine function of normal and mutant CFTRs in ER membranes. Such a system might be useful to examine interactions between CFTR and other proteins in the ER, including molecular chaperones, which might be important for CFTR function or normal intracellular processing. Demonstration that CFTR is active in the ER may suggest alternative therapies for CF, based on modifying CFTR function in this intracellular organelle.
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