CFTR: Ligand exchange between a permeant anion ([Au(CN)2] -) and an engineered cysteine (T338C) blocks the pore

José R. Serrano, Xuehong Liu, Erik R. Borg, Christopher S. Alexander, C. Frank Shaw, David C. Dawson

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1737-1748
Number of pages12
JournalBiophysical Journal
Volume91
Issue number5
DOIs
StatePublished - 2006

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Cystic Fibrosis Transmembrane Conductance Regulator
Anions
Cysteine
Ligands
Oocytes
Sulfhydryl Reagents
Chloride Channels
Mercaptoethanol
Dithiothreitol
Xenopus
Baths
Sulfhydryl Compounds

ASJC Scopus subject areas

  • Biophysics

Cite this

CFTR : Ligand exchange between a permeant anion ([Au(CN)2] -) and an engineered cysteine (T338C) blocks the pore. / Serrano, José R.; Liu, Xuehong; Borg, Erik R.; Alexander, Christopher S.; Shaw, C. Frank; Dawson, David C.

In: Biophysical Journal, Vol. 91, No. 5, 2006, p. 1737-1748.

Research output: Contribution to journalArticle

Serrano, José R. ; Liu, Xuehong ; Borg, Erik R. ; Alexander, Christopher S. ; Shaw, C. Frank ; Dawson, David C. / CFTR : Ligand exchange between a permeant anion ([Au(CN)2] -) and an engineered cysteine (T338C) blocks the pore. In: Biophysical Journal. 2006 ; Vol. 91, No. 5. pp. 1737-1748.
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