title: Interactions between permeant and blocking anions inside the CFTR chloride channel pore.
abstract: Binding of cytoplasmic anionic open channel blockers within the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is antagonized by extracellular Cl(-). In the present work, patch clamp recording was used to investigate the interaction between extracellular Cl(-) (and other anions) and cytoplasmic Pt(NO2)4(2-) ions inside the CFTR channel pore. In constitutively open (E1371Q-CFTR) channels, these different anions bind to two separate sites, located in the outer and inner vestibules of the pore respectively, in a mutually antagonistic fashion. A mutation in the inner vestibule (I344K) that greatly increased Pt(NO2)4(2-) binding affinity also greatly strengthened antagonistic Cl(-):blocker interactions as well as the voltage-dependence of block. Quantitative analysis of ion binding affinity suggested that the I344K mutation strengthened interactions not only with intracellular Pt(NO2)4(2-) ions but also with extracellular Cl(-), and that altered blocker Cl(-)- and voltage-dependence were due to the introduction of a novel type of antagonistic ion:ion interaction inside the pore that was independent of Cl(-) binding in the outer vestibule. It is proposed that this mutation alters the arrangement of anion binding sites inside the pore, allowing both Cl(-) and Pt(NO2)4(2-) to bind concurrently within the inner vestibule in a strongly mutually antagonistic fashion. However, the I344K mutation does not increase single channel conductance following disruption of Cl(-) binding in the outer vestibule in R334Q channels. Implications for the arrangement of ion binding sites in the pore, and their functional consequences for blocker binding and for rapid Cl(-) permeation, are discussed.
authors.affiliation: Department of Physiology & Biophysics, Dalhousie University, PO Box 15000, Halifax, Nova Scotia B3H 4R2, Canada. Electronic address: paul.linsdell@dal.ca.
authors.forename: Paul
authors.initials: P
authors.lastname: Linsdell
mesh_terms: D000465:Algorithms; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000838:Anions / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics; D002712:Chlorides / Q000378:metabolism* / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: D000596:Amino Acids; D000838:Anions; D002712:Chlorides; D050505:Mutant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator
keywords: Chloride channel; Cystic fibrosis transmembrane conductance regulator; Ion binding; Multi-ion pore behavior; Open channel block; Site-directed mutagenesis