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.