title: Asymmetric structure of the cystic fibrosis transmembrane conductance regulator chloride channel pore suggested by mutagenesis of the twelfth transmembrane region.
abstract: The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel contains 12 membrane-spanning regions which are presumed to form the transmembrane pore. Although a number of findings have suggested that the sixth transmembrane region plays a key role in forming the pore and determining its functional properties, the role of other transmembrane regions is currently not well established. Here we assess the functional importance of the twelfth transmembrane region, which occupies a homologous position in the carboxy terminal half of the CFTR molecule to that of the sixth transmembrane region in the amino terminal half. Five residues in potentially important regions of the twelfth transmembrane region were mutated individually to alanines, and the function of the mutant channels was examined using patch clamp recording following expression in mammalian cell lines. Three of the five mutations significantly weakened block of unitary Cl(-) currents by SCN(-), implying a partial disruption of anion binding within the pore. Two of these mutations also caused a large reduction in the steady-state channel mean open probability, suggesting a role for the twelfth transmembrane region in channel gating. However, in direct contrast to analogous mutations in the sixth transmembrane region, all mutants studied here had negligible effects on the anion selectivity and unitary Cl(-) conductance of the channel. The relatively minor effects of these five mutations on channel permeation properties suggests that, despite their symmetrical positions within the CFTR protein, the sixth and twelfth transmembrane regions make highly asymmetric contributions to the functional properties of the pore.
authors.affiliation: Department of Physiology & Biophysics, Dalhousie University, Halifax, Nova Scotia, B3H 4H7, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Gupta
authors.forename: A
authors.initials: A
authors.lastname: Evagelidis
authors.forename: J W
authors.initials: JW
authors.lastname: Hanrahan
authors.forename: P
authors.initials: P
authors.lastname: Linsdell
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000838:Anions; D016466:CHO Cells; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000235:genetics; D002712:Chlorides / Q000378:metabolism*; D006224:Cricetinae; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D004553:Electric Conductivity; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000235:genetics; D013861:Thiocyanates / Q000494:pharmacology; D014162:Transfection
chemical_list: D000838:Anions; C505032:CFTR protein, human; D002712:Chlorides; D010446:Peptide Fragments; D013861:Thiocyanates; D019005:Cystic Fibrosis Transmembrane Conductance Regulator