首页 | 本学科首页   官方微博 | 高级检索  
   检索      


Three charged amino acids in extracellular loop 1 are involved in maintaining the outer pore architecture of CFTR
Authors:Guiying Cui  Kazi S Rahman  Daniel T Infield  Christopher Kuang  Chengyu Z Prince  Nael A McCarty
Institution:1.Division of Pulmonary, Allergy and Immunology, Cystic Fibrosis, and Sleep, Department of Pediatrics, Center for Cystic Fibrosis and Airways Disease Research, Emory+Children’s Pediatric Research Center, Emory University School of Medicine and Children’s Healthcare of Atlanta, Atlanta, GA 30322;2.Parker H. Petit Institute for Bioengineering and Bioscience, and 3.School of Biology, Georgia Institute of Technology, Atlanta, GA 30332
Abstract:The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) bears six extracellular loops (ECL1–6); ECL1 is the site of several mutations associated with CF. Mutation R117H has been reported to reduce current amplitude, whereas D110H, E116K, and R117C/L/P may impair channel stability. We hypothesized that these amino acids might not be directly involved in ion conduction and permeation but may contribute to stabilizing the outer vestibule architecture in CFTR. We used cRNA injected oocytes combined with electrophysiological techniques to test this hypothesis. Mutants bearing cysteine at these sites were not functionally modified by extracellular MTS reagents and were blocked by GlyH-101 similarly to WT-CFTR. These results suggest that these three residues do not contribute directly to permeation in CFTR. In contrast, mutants D110R-, E116R-, and R117A-CFTR exhibited instability of the open state and significantly shortened burst duration compared with WT-CFTR and failed to be locked into the open state by AMP-PNP (adenosine 5′-(β,γ-imido) triphosphate); charge-retaining mutants showed mainly the full open state with comparably longer open burst duration. These interactions suggest that these ECL1 residues might be involved in maintaining the outer pore architecture of CFTR. A CFTR homology model suggested that E116 interacts with R104 in both the closed and open states, D110 interacts with K892 in the fully closed state, and R117 interacts with E1126 in the open state. These interactions were confirmed experimentally. The results suggest that D110, E116, and R117 may contribute to stabilizing the architecture of the outer pore of CFTR by interactions with other charged residues.
Keywords:
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号