Cystic fibrosis transmembrane conductance regulator: Using differential reactivity toward channel-permeant and channel-impermeant thiol-reactive probes to test a molecular model for the pore

Christopher Alexander, Anthony Ivetac, Xuehong Liu, Yohei Norimatsu, Jose R. Serrano, Allison Landstrom, Mark Sansom, David C. Dawson

Research output: Contribution to journalArticle

62 Citations (Scopus)

Abstract

The sixth transmembrane segment (TM6) of the CFTR chloride channel has been intensively investigated. The effects of amino acid substitutions and chemical modification of engineered cysteines (cysteine scanning) on channel properties strongly suggest that TM6 is a key component of the anion-conducting pore, but previous cysteine-scanning studies of TM6 have produced conflicting results. Our aim was to resolve these conflicts by combining a screening strategy based on multiple, thiol-directed probes with molecular modeling of the pore. CFTR constructs were screened for reactivity toward both channel-permeant and channel-impermeant thiol-directed reagents, and patterns of reactivity in TM6 were mapped onto two new, molecular models of the CFTR pore: one based on homology modeling using Sav1866 as the template and a second derived from the first by molecular dynamics simulation. Comparison of the pattern of cysteine reactivity with model predictions suggests that nonreactive sites are those where the TM6 side chains are occluded by other TMs. Reactive sites, in contrast, are generally situated such that the respective amino acid side chains either project into the predicted pore or lie within a predicted extracellular loop. Sites where engineered cysteines react with both channel-permeant and channel-impermeant probes occupy the outermost extent of TM6 or the predicted TM5-6 loop. Sites where cysteine reactivity is limited to channelpermeant probes occupy more cytoplasmic locations. The results provide an initial validation of two, new molecular models for CFTR and suggest that molecular dynamics simulation will be a useful tool for unraveling the structural basis of anion conduction by CFTR.

Original languageEnglish (US)
Pages (from-to)10078-10088
Number of pages11
JournalBiochemistry
Volume48
Issue number42
DOIs
StatePublished - Oct 27 2009

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Cystic Fibrosis Transmembrane Conductance Regulator
Molecular Models
Sulfhydryl Compounds
Cysteine
Molecular Dynamics Simulation
Anions
Molecular dynamics
Scanning
Amino Acids
Molecular Probes
Sulfhydryl Reagents
Chloride Channels
Molecular modeling
Chemical modification
Computer simulation
Amino Acid Substitution
Catalytic Domain
Screening
Substitution reactions

ASJC Scopus subject areas

  • Biochemistry

Cite this

Cystic fibrosis transmembrane conductance regulator : Using differential reactivity toward channel-permeant and channel-impermeant thiol-reactive probes to test a molecular model for the pore. / Alexander, Christopher; Ivetac, Anthony; Liu, Xuehong; Norimatsu, Yohei; Serrano, Jose R.; Landstrom, Allison; Sansom, Mark; Dawson, David C.

In: Biochemistry, Vol. 48, No. 42, 27.10.2009, p. 10078-10088.

Research output: Contribution to journalArticle

Alexander, Christopher ; Ivetac, Anthony ; Liu, Xuehong ; Norimatsu, Yohei ; Serrano, Jose R. ; Landstrom, Allison ; Sansom, Mark ; Dawson, David C. / Cystic fibrosis transmembrane conductance regulator : Using differential reactivity toward channel-permeant and channel-impermeant thiol-reactive probes to test a molecular model for the pore. In: Biochemistry. 2009 ; Vol. 48, No. 42. pp. 10078-10088.
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