Intracellular cysteines of the cystic fibrosis transmembrane conductance regulator (CFTR) modulate channel gating

Christian Ketchum, Hongwen Yue, Karen Alessi, Shreenivas Devidas, William Guggino, Peter Maloney

Research output: Contribution to journalArticle

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ATP-binding cassette superfamily, is a cAMP-activated chloride channel. CFTR contains two transmembrane domains (TMDs), two nucleotide-binding domains (NBDs), and a regulatory (R) domain. We found that whole-cell CFTR-dependent Cl - currents in Xenopus laevis oocytes were sensitive to HgCl 2 , suggesting that modification of endogenous cysteines alters channel activity. To understand better this phenomenon, site-directed mutagenesis was employed to generate both individual cysteine replacements and a version of the molecule with no cysteines in the hydrophobic sector. Each mutant displayed a forskolin/IBMX-activated Cl-conductance similar to wild type, indicating that none of the cysteines located within the TMDs is essential. Subsequent single-channel analysis of inside-out patches excised from HEK293 cells expressing either cysteine-less or wild-type CFTR showed that intracellular application of a membrane impermeant sulphydryl reagent, p-chloromercuribenzosulfonate (PCMBS), significantly reduced open probability without affecting ion selectivity or conductance. The cysteine-less molecule also acquired a voltage-dependent sensitivity to extracellular PCMBS not observed in the wild type, perhaps due to a more flexible conformation that allowed PCMBS access to the intracellular surface. Together, these experiments suggest that endogenous intracellular cysteines, located primarily within the NBDs and/or R domain, influence channel gating.

Original languageEnglish (US)
Pages (from-to)1-8
Number of pages8
JournalCellular Physiology and Biochemistry
Volume12
Issue number1
DOIs
StatePublished - Jan 1 2002

Keywords

  • ABC transporter
  • ATP-binding cassette
  • CFTR
  • Channel gating
  • Cysteine mutagenesis
  • Open probability
  • Sulphydryl labeling

ASJC Scopus subject areas

  • Physiology

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