Cysteine accessibility in ClC-0 supports conservation of the ClC intracellular vestibule.
ClC chloride channels, which are ubiquitously expressed in mammals, have a unique double-barreled structure, in which each monomer forms its own pore. Identification of pore-lining elements is important for understanding the conduction properties and unusual gating mechanisms of these channels. Structures of prokaryotic ClC transporters do not show an open pore, and so may not accurately represent the open state of the eukaryotic ClC channels. In this study we used cysteine-scanning mutagenesis and modification (SCAM) to screen >50 residues in the intracellular vestibule of ClC-0. We identified 14 positions sensitive to the negatively charged thiol-modifying reagents sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) or sodium 4-acetamido-4'-maleimidylstilbene-2'2-disulfonic acid (AMS) and show that 11 of these alter pore properties when modified. In addition, two MTSES-sensitive residues, on different helices and in close proximity in the prokaryotic structures, can form a disulfide bond in ClC-0. When mapped onto prokaryotic structures, MTSES/AMS-sensitive residues cluster around bound chloride ions, and the correlation is even stronger in the ClC-0 homology model developed by Corry et al. (2004). These results support the hypothesis that both secondary and tertiary structures in the intracellular vestibule are conserved among ClC family members, even in regions of very low sequence similarity.
Engh
Anita M
AM
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, CA 94305, USA.
Maduke
Merritt
M
D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D001369:Axons / Q000502:physiology; D002352:Carrier Proteins / Q000378:metabolism; D018118:Chloride Channels / Q000187:drug effects* / Q000235:genetics; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry / Q000494:pharmacology*; D004220:Disulfides / Q000737:chemistry; D007202:Indicators and Reagents; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D008564:Membrane Potentials / Q000502:physiology; D008698:Mesylates / Q000494:pharmacology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009153:Mutagens / Q000494:pharmacology; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013267:Stilbenes / Q000494:pharmacology; D013451:Sulfonic Acids / Q000494:pharmacology; D014981:Xenopus
D002352:Carrier Proteins; D018118:Chloride Channels; D003432:Cross-Linking Reagents; D004220:Disulfides; D007202:Indicators and Reagents; D008698:Mesylates; D009153:Mutagens; D013267:Stilbenes; D013451:Sulfonic Acids; C079532:4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid; C109153:(2-sulfonatoethyl)methanethiosulfonate; D003545:Cysteine