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

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 100 nM [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.