CFTR: Ligand exchange between a permeant anion ([Au(CN)2]-) and an engineered cysteine (T338C) blocks the pore
| Autor: | Jose R. Serrano, C. Frank Shaw, Xuehong Liu, David C. Dawson, Erik R. Borg, Christopher Alexander |
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| Rok vydání: | 2006 |
| Předmět: |
Anions
Cell Membrane Permeability Inorganic chemistry Biophysics Cystic Fibrosis Transmembrane Conductance Regulator Ligands Protein Engineering Dithiothreitol chemistry.chemical_compound Structure-Activity Relationship Xenopus laevis Animals Cysteine Channels Receptors and Electrical Signaling Cells Cultured Cyanates chemistry.chemical_classification Cyanides biology Ligand Electric Conductivity Conductance Cystic fibrosis transmembrane conductance regulator Gold Compounds Crystallography chemistry Amino Acid Substitution Reagent Thiol Chloride channel biology.protein Mutagenesis Site-Directed Oocytes Gold Ion Channel Gating |
| Zdroj: | Biophysical journal. 91(5) |
| ISSN: | 0006-3495 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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