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.
Serrano
José R
JR
Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, Oregon 97239, USA.
Liu
Xuehong
X
Borg
Erik R
ER
Alexander
Christopher S
CS
Shaw
C Frank
CF
Dawson
David C
DC
D019943:Amino Acid Substitution; D000818:Animals; D000838:Anions; D002463:Cell Membrane Permeability / Q000502:physiology*; D002478:Cells, Cultured; D003485:Cyanates; D003486:Cyanides / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000378:metabolism*; D004553:Electric Conductivity; D006046:Gold; D017612:Gold Compounds / Q000378:metabolism*; D015640:Ion Channel Gating; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology*; D015202:Protein Engineering / Q000379:methods; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
D000838:Anions; D003485:Cyanates; D003486:Cyanides; D017612:Gold Compounds; D008024:Ligands; C043041:gold cyanide; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006046:Gold; D003545:Cysteine