title: Functional differences in pore properties between wild-type and cysteine-less forms of the CFTR chloride channel.
abstract: Studies of the structure and function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel have been advanced by the development of functional channel variants in which all 18 endogenous cysteine residues have been mutated ("cys-less" CFTR). However, cys-less CFTR has a slightly higher single-channel conductance than wild-type CFTR, raising questions as to the suitability of cys-less as a model of the wild-type CFTR pore. We used site-directed mutagenesis and patch-clamp recording to investigate the origin of this conductance difference and to determine the extent of functional differences between wild-type and cys-less CFTR channel permeation properties. Our results suggest that the conductance difference is the result of a single substitution, of C343: the point mutant C343S has a conductance similar to cys-less, whereas the reverse mutation, S343C in a cys-less background, restores wild-type conductance levels. Other cysteine substitutions (C128S, C225S, C376S, C866S) were without effect. Substitution of other residues for C343 suggested that conductance is dependent on amino acid side chain volume at this position. A range of other functional pore properties, including interactions with channel blockers (Au[CN]                            (2)                                      (-)                         , 5-nitro-2-[3-phenylpropylamino]benzoic acid, suramin) and anion permeability, were not significantly different between wild-type and cys-less CFTR. Our results suggest that functional differences between these two CFTR constructs are of limited scale and scope and result from a small change in side chain volume at position 343. These results therefore support the use of cys-less as a model of the CFTR pore region.
authors.affiliation: Department of Physiology and Biophysics, Dalhousie University, 5850 College Street, Halifax, NS B3H 1X5, Canada.
authors.forename: Ryan G
authors.initials: RG
authors.lastname: Holstead
authors.forename: Man-Song
authors.initials: MS
authors.lastname: Li
authors.forename: Paul
authors.initials: P
authors.lastname: Linsdell
mesh_terms: D000818:Animals; D000838:Anions / Q000737:chemistry; D002478:Cells, Cultured; D006224:Cricetinae; D003545:Cysteine / Q000235:genetics*; D003550:Cystic Fibrosis / Q000235:genetics / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010539:Permeability
chemical_list: D000838:Anions; D050505:Mutant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D003545:Cysteine