title: Cytoplasmic pathway followed by chloride ions to enter the CFTR channel pore.
abstract: Most ATP-binding cassette (ABC) proteins function as ATP-dependent membrane pumps. One exception is the cystic fibrosis transmembrane conductance regulator (CFTR), an ABC protein that functions as a Cl(-) ion channel. As such, the CFTR protein must form a continuous pathway for the movement of Cl(-) ions from the cytoplasm to the extracellular solution when in its open channel state. Extensive functional investigations have characterized most parts of this Cl(-) permeation pathway. However, one region remains unexplored-the pathway connecting the cytoplasm to the membrane-spanning pore. We used patch clamp recording and extensive substituted cysteine accessibility mutagenesis to identify amino acid side-chains in cytoplasmic regions of CFTR that lie close to the pathway taken by Cl(-) ions as they pass from the cytoplasm through this pathway. Our results suggest that Cl(-) ions enter the permeation pathway via a single lateral tunnel formed by the cytoplasmic parts of the protein, and then follow a fairly direct central pathway towards the membrane-spanning parts of the protein. However, this pathway is not lined continuously by any particular part of the protein; instead, the contributions of different cytoplasmic regions of the protein appear to change as the permeation pathway approaches the membrane, which appears to reflect the ways in which different cytoplasmic regions of the protein are oriented towards its central axis. Our results allow us to define for the first time the complete Cl(-) permeation pathway in CFTR, from the cytoplasm to the extracellular solution.
authors.affiliation: Department of Physiology and Biophysics, Dalhousie University, PO Box 15000, Halifax, NS, B3H 4R2, Canada.
authors.forename: Yassine
authors.initials: Y
authors.lastname: El Hiani
authors.affiliation: Department of Physiology and Biophysics, Dalhousie University, PO Box 15000, Halifax, NS, B3H 4R2, Canada.
authors.forename: Alexander
authors.initials: A
authors.lastname: Negoda
authors.affiliation: Department of Physiology and Biophysics, Dalhousie University, PO Box 15000, Halifax, NS, B3H 4R2, Canada. paul.linsdell@dal.ca.
authors.forename: Paul
authors.initials: P
authors.lastname: Linsdell
mesh_terms: D000200:Action Potentials / Q000187:drug effects; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D020134:Catalytic Domain; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism*; D006224:Cricetinae; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D007477:Ions / Q000737:chemistry / Q000378:metabolism; D016296:Mutagenesis; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary
chemical_list: D002712:Chlorides; D007477:Ions; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases
keywords: Chloride channel; Cystic fibrosis transmembrane conductance regulator; Cytoplasmic loop; Pore; Substituted cysteine accessibility mutagenesis