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Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein
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Localizing a gate in CFTR.

Xiaolong Gao1, Tzyh-Chang Hwang2

  • 1Dalton Cardiovascular Research Center and Departments of Biological Engineering and.

Proceedings of the National Academy of Sciences of the United States of America
|February 13, 2015
PubMed
Summary

The cystic fibrosis transmembrane conductance regulator (CFTR) has a gate located in its narrow pore region (residues 337-344 in TM6), distinct from other ABC transporters. This gate controls chloride ion flow.

Keywords:
ABC transportersanion channelscystic fibrosisgating

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Area of Science:

  • Biophysics
  • Molecular Biology
  • Ion Channel Function

Background:

  • Previous studies depicted the CFTR chloride permeation pathway as a narrow tunnel with vestibules.
  • The precise location of CFTR's gate(s) remained undetermined.
  • Transmembrane segments (TMs) were identified as pore-lining, but gate function was unclear.

Purpose of the Study:

  • To pinpoint the location of the gate in the CFTR ion channel.
  • To investigate the gating mechanism of CFTR using a channel-permeant probe.
  • To differentiate CFTR gating from other ATP-binding cassette (ABC) transporters.

Main Methods:

  • Utilized a channel-permeant probe, [Au(CN)2](-), for site-specific modification.
  • Introduced cysteine residues at various positions within the CFTR pore.
  • Assessed probe accessibility in different functional states (open/closed) and in mutated CFTR variants (G1349D).

Main Results:

  • Cysteines on the cytoplasmic side of the predicted narrow region (TM6 residues 344, TM12 residue 1148) were accessible in both open and closed states.
  • Cysteines external to the narrow region (TM6 residues 334, 335, 337) were not reactive to cytoplasmic [Au(CN)2](-) in the absence of ATP.
  • These external cysteines showed increased accessibility to extracellular [Au(CN)2](-) when CFTR open probability was reduced (G1349D mutation).

Conclusions:

  • CFTR possesses a gate located between residues 337 and 344 in TM6, within the predicted narrow pore segment.
  • This gate region likely also functions as the chloride ion selectivity filter.
  • CFTR's gate position is unique among ABC transporters, distinguishing its mechanism of ion translocation.