Interaction between 2 extracellular loops influences the activity of the cystic fibrosis transmembrane conductance regulator chloride channel

Abstract

Activity of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is thought to be controlled by cytoplasmic factors. However, recent evidence has shown that overall channel activity is also influenced by extracellular anions that interact directly with the extracellular loops (ECLs) of the CFTR protein. Very little is known about the structure of the ECLs or how substances interacting with these ECLs might affect CFTR function. We used patch-clamp recording to investigate the accessibility of cysteine-reactive reagents to cysteines introduced throughout ECL1 and 2 key sites in ECL4. Furthermore, interactions between ECL1 and ECL4 were investigated by the formation of disulfide crosslinks between cysteines introduced into these 2 regions. Crosslinks could be formed between R899C (in ECL4) and a number of sites in ECL1 in a manner that was dependent on channel activity, suggesting that the relative orientation of these 2 loops changes on activation. Formation of these crosslinks inhibited channel function, suggesting that relative movement of these ECLs is important to normal channel function. Implications of these findings for the effects of mutations in the ECLs that are associated with cystic fibrosis and interactions with extracellular substances that influence channel activity are discussed.

The cystic fibrosis transmembrane conductance regulator is an extracellular chloride sensor

“…Interestingly, a recent study by Cui et al [ 13 ] has shown that three charged amino acid residues in ECL1 (D110, E116 and R117) are involved in stabilising the architecture of the outer pore of CFTR by interacting with other charged residues, including K892 in ECL4. Furthermore, our lab has recently demonstrated, through cross-linking of residues in ECL1 and ECL4, that the relative movement of these two ECLs is necessary for normal channel gating [ 10 ] which further demonstrates the important role of the ECLs in CFTR channel function.…”

Structure-Function Relationships of CFTR in Health and Disease: The Pancreas Story

“…These mutations affect the stability of the CFTR ion pore, resulting in reduced conductance of CFTR (34,42). Q890X and K892C mutations in ECL4 have been reported to affect channel gating and extracellular anion interaction (10,29,42). ECL4 is the only extracellular loop that contains Nlinked glycosylation sites (N894 and N900) (10).…”

“…Q890X and K892C mutations in ECL4 have been reported to affect channel gating and extracellular anion interaction (10,29,42). ECL4 is the only extracellular loop that contains Nlinked glycosylation sites (N894 and N900) (10). Many studies have been performed on the function of CFTR's intracytoplasmic loops (ICLs) including their roles in regulating inter-molecular interactions as well as CFTR interactions with other proteins.…”