Channel-lining residues in the M3 membrane-spanning segment of the cystic fibrosis transmembrane conductance regulator.
The cystic fibrosis transmembrane conductance regulator (CFTR) forms a chloride-selective channel. Residues from the 12 putative membrane-spanning segments form at least part of the channel lining. We need to identify the channel-lining residues in order to understand the structural basis for the channel's functional properties. Using the substituted-cysteine-accessibility method we mutated to cysteine, one at a time, 24 consecutive residues (Asp192-Ile215) in the M3 membrane-spanning segment. Cysteines substituted for His199, Phe200, Trp202, Ile203, Pro205, Gln207, Leu211, and Leu214 reacted with charged, sulfhydryl-specific reagents that are derivatives of methanethiosulfonate (MTS). We infer that these residues are on the water-accessible surface of the protein and probably form a portion of the channel lining. When plotted on an alpha-helical wheel the exposed residues from Gln207 to Leu214 lie within an arc of 60 degrees; the exposed residues in the cytoplasmic half (His199-Ile203) lie within an arc of 160 degrees. We infer that the secondary structures of the extracellular and cytoplasmic halves of M3 are alpha-helical and that Pro205, in the middle of the M3 segment, may bend the M3 segment, moving the cytoplasmic end of the segment in toward the central axis of the channel. The bend in the M3 segment may help to narrow the channel lumen near the cytoplasmic end. In addition, unlike full-length CFTR, the current induced by the deletion construct, Delta259, is inhibited by the MTS reagents, implying that the channel structure of Delta259 is different than the channel structure of wild-type CFTR.
Center for Molecular Recognition, Department of Physiology, College of Physicians and Surgeons, Columbia University, New York 10032, USA.
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D000818:Animals; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D005822:Genetic Vectors / Q000096:biosynthesis / Q000187:drug effects; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000235:genetics; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D008698:Mesylates / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009865:Oocytes; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D017433:Protein Structure, Secondary; D017384:Sequence Deletion; D014867:Water; D014982:Xenopus laevis
C505032:CFTR protein, human; D018118:Chloride Channels; D008565:Membrane Proteins; D008698:Mesylates; D010446:Peptide Fragments; C093171:methanethiosulfonate ethylammonium; D014867:Water; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C109153:(2-sulfonatoethyl)methanethiosulfonate; D005020:Ethyl Methanesulfonate