Non-pore lining amino acid side chains influence anion selectivity of the human CFTR Cl- channel expressed in mammalian cell lines.
1. The effects of individually mutating two adjacent threonine residues in the sixth membrane-spanning region (TM6) of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel on permeation properties were examined using patch clamp recording from mammalian cell lines stably expressing human CFTR. 2. A number of mutations of T338 significantly affected the permeation properties of the channel. Increases and decreases in single channel conductance were observed for different mutants. Anion selectivity was strongly affected, with no two channel variants sharing the same selectivity sequence. Several mutations led to strong inward rectification of the macroscopic current-voltage relationship. The effects of these mutations on permeation properties were correlated with the size of the amino acid side chain substituted, rather than its chemical nature. 3. Most mutations of T339 resulted in a lack of functional channel expression and apparent misprocessing of the protein. One mutant, T339V, was characterized in detail; its permeation properties were significantly altered, although these effects were not as strong as for T338 mutations. 4. These results suggest an important role for T338 in controlling the permeation properties of the CFTR Cl- channel. It is suggested that mutation of this residue alters the interaction between permeating anions and the channel pore via an indirect effect on the orientation of the TM6 helix.
Linsdell
P
P
Department of Physiology, McGill University, Montreal, Quebec, Canada H3G 1Y6. linsdell@physio.mcgill.ca
Zheng
S X
SX
Hanrahan
J W
JW
D019943:Amino Acid Substitution; D000818:Animals; D000838:Anions; D016466:CHO Cells; D002460:Cell Line; D002463:Cell Membrane Permeability; D006224:Cricetinae; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000096:biosynthesis / Q000737:chemistry* / Q000502:physiology*; D006801:Humans; D007668:Kidney; D008322:Mammals; D008564:Membrane Potentials; D008727:Methotrexate / Q000494:pharmacology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D000838:Anions; C505032:CFTR protein, human; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D008727:Methotrexate