CFTR: Ligand Exchange between a Permeant Anion ([Au(CN)2]−) and an Engineered Cysteine (T338C) Blocks the Pore

• Published in: Biophysical journal 2006-09, Vol.91 (5), p.1737-1748
• Main Authors: Serrano, José R., Liu, Xuehong, Borg, Erik R., Alexander, Christopher S., Shaw, C. Frank, Dawson, David C.
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Animals; Anions; Cell Membrane Permeability - physiology; Cells, Cultured; Channels, Receptors, and Electrical Signaling; Cyanates; Cyanides - metabolism; Cysteine - metabolism; Cystic Fibrosis Transmembrane Conductance Regulator - chemistry; Cystic Fibrosis Transmembrane Conductance Regulator - metabolism; Electric Conductivity; Gold; Gold Compounds - metabolism; Ion Channel Gating; Ligands; Mutagenesis, Site-Directed; Oocytes - physiology; Protein Engineering - methods; Structure-Activity Relationship; Xenopus laevis
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1529/biophysj.105.078899

Previous attempts to identify residues that line the pore of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have utilized cysteine-substituted channels in conjunction with impermeant, thiol-reactive reagents like MTSET+ and MTSES−. We report here that the permeant, pseudohalide anion [Au(CN)2]− can also react with a cysteine engineered into the pore of the CFTR channel. Exposure of Xenopus oocytes expressing the T338C CFTR channel to as little as 100nM [Au(CN)2]− produced a profound reduction in conductance that was not reversed by washing but was reversed by exposing the oocytes to a competing thiol like DTT (dithiothreitol) and 2-ME (2-mercaptoethanol). In detached, inside out patches single-channel currents were abolished by [Au(CN)2]− and activity was not restored by washing [Au(CN)2]− from the bath. Both single-channel and macroscopic currents were restored, however, by exposing [Au(CN)2]−-blocked channels to excess [CN]−. The results are consistent with the hypothesis that [Au(CN)2]− can participate in a ligand exchange reaction with the cysteine thiolate at 338 such that the mixed-ligand complex, with a charge of −1, blocks the anion conduction pathway.