title: Intracellular cysteines of the cystic fibrosis transmembrane conductance regulator (CFTR) modulate channel gating.
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.
authors.affiliation: Department of Physiology, Johns Hopkins University School of Medicine, Baltimore MD 21205, USA.
authors.forename: Christian J
authors.initials: CJ
authors.lastname: Ketchum
authors.forename: Hongwen
authors.initials: H
authors.lastname: Yue
authors.forename: Karen A
authors.initials: KA
authors.lastname: Alessi
authors.forename: Shreenivas
authors.initials: S
authors.lastname: Devidas
authors.forename: William B
authors.initials: WB
authors.lastname: Guggino
authors.forename: Peter C
authors.initials: PC
authors.lastname: Maloney
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D003545:Cysteine / Q000187:drug effects / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014162:Transfection; D014982:Xenopus laevis
chemical_list: C505032:CFTR protein, human; D002712:Chlorides; D010726:Phosphodiesterase Inhibitors; D013439:Sulfhydryl Reagents; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D003545:Cysteine; D015056:1-Methyl-3-isobutylxanthine