title: Positioning of extracellular loop 1 affects pore gating of the cystic fibrosis transmembrane conductance regulator.
abstract: The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is a chloride ion channel, the dysfunction of which directly leads to the life-shortening disease CF. Extracellular loop 1 (ECL1) of CFTR contains several residues involved in stabilizing the open state of the channel; some, including D110, are sites of disease-associated gating mutations. Structures from related proteins suggest that the position of CFTR's extracellular loops may change considerably during gating. To better understand the roles of ECL1 in CFTR function, we utilized functional cysteine cross-linking to determine the effects of modulation of D110C-CFTR and of a double mutant of D110C with K892C in extracellular loop 4 (ECL4). The reducing agent DTT elicited a large potentiation of the macroscopic conductance of D110C/K892C-CFTR, likely due to breakage of a spontaneous disulfide bond between C110 and C892. DTT-reduced D110C/K892C-CFTR was rapidly inhibited by binding cadmium ions with high affinity, suggesting that these residues frequently come in close proximity in actively gating channels. Effects of DTT and cadmium on modulation of pore gating were demonstrated at the single-channel level. Finally, disulfided D110C/K892C-CFTR channels were found to be less sensitive than wild-type or DTT-treated D110C/K892C-CFTR channels to stimulation by IBMX, suggesting an impact of this conformational restriction on channel activation by phosphorylation. The results are best explained in the context of a model of CFTR gating wherein stable channel opening requires correct positioning of functional elements structurally influenced by ECL1.
authors.affiliation: Division of Pulmonology, 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, Georgia; and Graduate Division of Biological and Biomedical Sciences, Emory University, Atlanta, Georgia.
authors.forename: Daniel T
authors.initials: DT
authors.lastname: Infield
authors.affiliation: Division of Pulmonology, 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, Georgia; and.
authors.forename: Guiying
authors.initials: G
authors.lastname: Cui
authors.affiliation: Division of Pulmonology, 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, Georgia; and.
authors.forename: Christopher
authors.initials: C
authors.lastname: Kuang
authors.affiliation: Division of Pulmonology, 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, Georgia; and namccar@emory.edu.
authors.forename: Nael A
authors.initials: NA
authors.lastname: McCarty
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D002478:Cells, Cultured; D018118:Chloride Channels / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology; D018408:Patch-Clamp Techniques / Q000379:methods; D013329:Structure-Activity Relationship
chemical_list: D018118:Chloride Channels; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000255:Adenosine Triphosphate; D003545:Cysteine
keywords: ATP-binding cassette transporter; chloride channel; cysteine-mediated cross-linking; cystic fibrosis transmembrane conductance regulator; structure-function