F508del CFTR with two altered RXR motifs escapes from ER quality control but its channel activity is thermally sensitive.
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.
Hegedus
Tamás
T
Department of Biochemistry and Biophysics and Cystic Fibrosis T&R Center, UNC, 5011 Thurston-Bowles Bldg., Chapel Hill, NC 27510-7248, USA.
Aleksandrov
Andrei
A
Cui
Liying
L
Gentzsch
Martina
M
Chang
Xiu-Bao
XB
Riordan
John R
JR
D020816:Amino Acid Motifs; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D009154:Mutation; D011487:Protein Conformation; D047488:Retinoid X Receptors / Q000737:chemistry* / Q000378:metabolism; D013696:Temperature
D047488:Retinoid X Receptors; D019005:Cystic Fibrosis Transmembrane Conductance Regulator