Most cystic fibrosis (CF) patients carry the F508del mutation in the CFTR chloride
channel protein resulting in its misassembly, retention in the endoplasmic reticulum
(ER), and proteasomal degradation. Therefore, characterization of the retention and
attempts to rescue the mutant CFTR are a major focus of CF research. Earlier, we had
shown that four arginine-framed tripeptide (AFT) signals in CFTR participate in the
quality control. Now we have mutated these four AFTs in all possible combinations
and found that simultaneous inactivation of two of them (R29K and R555K) is necessary
and sufficient to overcome F508del CFTR retention. Immunofluorescence staining of
BHK cells expressing this variant indicates that it matures and is routed to the plasma
membrane. Acquisition of at least some wild-type structure was detected in the pattern
of proteolytic digestion fragments. Functional activity at the cell surface was evident
in chloride efflux assays. However, single channel activity of the rescued mutant
measured in planar lipid bilayers diminished as temperature was increased from 30
to 37 degrees C. These findings support the idea that absence of Phe 508 causes not
only a kinetic folding defect but also steady-state structural instability. Therefore
effective molecular therapies developed to alleviate disease caused by F508del and
probably other misprocessing mutants will require overcoming both their kinetic and
steady-state impacts.
