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© 1993 Oxford University Press

RESEARCH-ARTICLE

Molecular basis of defective anion transport in L cells expressing recombinant forms of CFTR

Yiping Yang1,+, Daniel C. Devor5, John F. Engelhardt1,+, Stephen A. Ernst2, Theresa V. Strong3, Francis S. Collins1,3, Jonathan A. Cohn4, Raymond A. Frizzell5 and James M. Wilson1,+,*

1Department of Internal Medicine, Biological Chemistry, University of Michigan Ann Arbor, MI 48109 2Department of Anatomy and Cell Biology, University of Michigan Ann Arbor, MI 48109 3Department of Human Genetics, University of Michigan Ann Arbor, MI 48109 4Department of Medicine, Duke University and VA Medical Center Durham, NC 27710 5Department of Physiology and Biophysics, University of Alabama at Birmingham Birmingham, AL 35294, USA

*To whom correspondence should be addressed

Received February 5, 1993; Revised June 14, 1993; Accepted June 14, 1993

Cystic fibrosis (CF) is caused by mutations in the gene encoding a chloride channel called the CF transmembrane conductance regulator (CFTR). A single mutation in this gene, deletion of three nucleotides that leads to the absence of phenylalanine 508 (i.e., {Delta}F508), is found on 70% of all CF chromosomes. To explore the molecular mechanism(s) responsible for defective chloride transport in patients with CF, we have studied the processing, localization, and function of wild type (W.T.), {Delta}F508 and G551D CFTR (a G->D missense mutation at position 551) in retrovirus transduced L cells. Cell transduced with W.T. CFTR expressed a 170 kd CFTR protein that was endoglycosidase H (Endo H) resistant, localized to the plasma membrane, and generated a cAMP-mediated anion conductance (GCl) when stimulated with standard concentrations of forskolin (5 µM), cpt cAMP (400 µM) and IBMX (100 µM). The G551D CFTR was indistinguishable from W.T. CFTR with respect to post-translational processing and localization, but it did not produce a cAMP-activated GCI in response to the standard stimulation cocktail. However, raising the IBMX concentration to 4 mM produced Gc, in G551D expressing cells. Cells transduced with {Delta}F508 CFTR expressed an Endo H sensitive CFTR protein (~140 kd) that was found in a cytosolic, perinuclear location. These cells did not respond to the standard cocktail, but ~20% of cells increased GCI when the cocktail contained 4 mM IBMX. Incubation of cells at 26°C for 48 hours prior to analysis elicited responses in {Delta}F508 expressing cells at low IBMX concentrations, but had no effect on the responses of cells expressing W.T. or G551D CFTR. The response of {Delta}F508 to 26°C was associated with plasma membrane localization of CFTR protein. These results suggest that there are two mechanisms whereby CFTR mutations lead to loss of cAMP-responsive GCI. First, shown by G551D CFTR, the protein can be processed and targeted to the plasma membrane correctly, but lack full responsiveness to stimulation by cAMP. Second, as examplified by {Delta}F508 CFTR, a partially functional protein which is not targeted to its correct cellular location can also lead to loss of the cAMP, responsive GCI.

+present address: Institute for Human Gene Therapy, University of Pennsylvania Medical Center, The Wistar Institute, Room 204, 36th and Spruce Streets, Philadelphia, PA 19104, USA


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