Amino acid residues lining the chloride channel of the cystic fibrosis transmembrane conductance regulator.
The cystic fibrosis transmembrane conductance regulator forms a chloride channel that is regulated by phosphorylation and intracellular ATP levels. The structure of the channel-forming domains is undetermined. To identify the residues lining this channel we substituted cysteine, one at a time, for 9 consecutive residues (91-99) in the M1 membrane-spanning segment. The cysteine substitution mutants were expressed in Xenopus oocytes. We determined the accessibility of the engineered cysteine to charged, sulfhydryl-specific methanethiosulfonate reagents added extracellularly. We assume that, among residues in membrane-spanning segments, only those lining the channel will be accessible to react with these hydrophilic reagents and that such a reaction would irreversibly alter conduction through the channel. Only the cysteines substituted for Gly-91, Lys-95, and Gln-98 were accessible to the reagents. We conclude that these residues are in the channel lining. The periodicity of these residues is consistent with an alpha-helical secondary structure.
Akabas
M H
MH
Center for Molecular Recognition, Columbia University, New York, New York 10032.
Kaufmann
C
C
Cook
T A
TA
Archdeacon
P
P
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D001483:Base Sequence; D018118:Chloride Channels / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D003550:Cystic Fibrosis; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D018076:DNA, Complementary; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D014982:Xenopus laevis
D000596:Amino Acids; C505032:CFTR protein, human; D018118:Chloride Channels; D018076:DNA, Complementary; D008565:Membrane Proteins; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator