CFTR plays a critical role in chloride transport across airway epithelial cells. Loss of function mutations in this gene result in cystic fibrosis (CF). Because of the high carrier frequency of the gene encoding CFTR and the severe disease caused by mutations in this gene, this protein has been the focus of extensive study. It is arguably the best understood membrane protein associated with human disease. CFTR belongs to the ATP binding cassette (ABC) transporter superfamily and consists of two membrane spanning domains, two nucleotide binding domains (NBD1, NBD2) and a unique R region. Ninety percent of CF patients carry at least one CFTR allele with a three base pair deletion that results in the absence of a phenylalanine (F) in the first nucleotide binding domain of the CFTR protein. This ?F508 mutation results in misfolding of the protein, targeting it for proteosomal degradation. Little of the mutated protein reaches the cell surface. Experimental conditions that allow transport of the mutant protein to the cell surface reveal that the loss of F508 from NBD1 has additional consequences. Electrophysiological studies showed that ?F508-CFTR has much greater closed times, resulting in a large decrease in open probability, thus altering its capacity to transport Cl- across the epithelial surface. The stability of the small amount of the mutant protein that reaches the cells surface is also compromised, resulting in a shortened cell surface half life. Characterization of the biochemical basis for retention of CFTR in the ER and attempts to improve stability and transport of the mutant CFTR are important for identifying new strategies for the treatment of CF. In addition, information concerning CFTR transport may also aid in the treatment of other diseases caused by deficiencies in protein trafficking and altered protein folding. A number of studies have examined the effects of physical conditions, small molecules and second site mutations on the trafficking and stability of the ?F508 protein. A major limitation in the interpretation of these studies is that most have been carried out in vitro, many using non polarized epithelial cell lines. In most cases, it is unknown whether the information gleaned from these in vitro experiments will translate directly to the transport and function of ?F508-CFTR in vivo. Furthermore, it is uncertain if the maneuvers that increase ?F508 -CFTR function in vitro will result in a similar increase in apical ?F508 CFTR in vivo and, if so, whether this will result in sufficient CFTR function to be of physiologic consequence to the organism. In this application we address this limitation. We propose to develop an animal model that will allow the effects of both chemical interventions and second site mutations on ?F508-CFTR transport to be tested rapidly in vivo. PUBLIC HEALTH: Cystic fibrosis is a common, severe, genetic disease with a carrier frequency of approximately 1/2500 in Caucasians. It is caused by mutations in a gene name CFTR. In 90% of individuals a three base pair deletion, which results in the loss of a single amino acid (?F508 mutation), leads to the development of disease. Morbidity and death are primarily the result of loss of lung function secondary to the accumulation of viscous mucus in the lung and subsequent colonization of the airways with microorganisms. Recently studies carried out in vitro in cell lines suggest that it may be possible to treat this disease with small molecules that improve the function of the CFTR protein with the ?F508 mutation. In this application we propose to develop models that will not only test the feasibility of this approach, but that can also be used for the rapid screening for such therapeutic agents.
Cystic fibrosis is a common, severe, genetic disease with a carrier frequency of approximately 1/2500 in Caucasians. It is caused by mutations in a gene name CFTR. In 90% of individuals a three base pair deletion, which results in the loss of a single amino acid ( ?F508 mutation), leads to the development of disease. Morbidity and death are primarily the result of loss of lung function secondary to the accumulation of viscous mucus in the lung and subsequent colonization of the airways with microorganisms. Recently studies carried out in vitro in cell lines suggest that it may be possible to treat this disease with small molecules that improve the function of the CFTR protein with the ?F508 mutation. In this application we propose to develop models that will not only test the feasibility of this approach, but that can also be used for the rapid screening for such therapeutic agents.
