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Author Notes:

Correspondence to Nael A. McCarty: Email: namccar@emory.edu.

G. Cui designed and carried out the electrophysiological measurements, analyzed data, prepared the figures, and wrote the manuscript.

D.T. Infield was involved in designing experiments and contributed to manuscript preparation.

C. Kuang and C.Z. Prince made all of the DNA constructs.

K.S. Rahman made the CFTR homology model and predicted the possible amino acids pairs for the paper.

C. Kuang performed surgery and prepared Xenopus oocytes.

N.A. McCarty planned the project, designed experiments, and contributed to the manuscript preparation.

Merritt C. Maduke served as editor.

The authors declare no competing financial interests.

Subjects:

Research Funding:

This research was supported by research grant R01-DK056481 from the National Institutes of Health to N.A. McCarty.

Keywords:

  • Science & Technology
  • Life Sciences & Biomedicine
  • Physiology
  • TRANSMEMBRANE CONDUCTANCE REGULATOR
  • CHANNEL VOLTAGE SENSOR
  • BETA-SCORPION TOXIN
  • CYSTIC-FIBROSIS
  • CHLORIDE CHANNEL
  • POSITIVE CHARGES
  • CATALYTIC CYCLE
  • ATP HYDROLYSIS
  • P-GLYCOPROTEIN
  • CL-CHANNELS

Three charged amino acids in extracellular loop 1 are involved in maintaining the outer pore architecture of CFTR

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Journal Title:

Journal of General Physiology

Volume:

Volume 144, Number 2

Publisher:

, Pages 159-179

Type of Work:

Article | Final Publisher PDF

Abstract:

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) bears six extracellular loops (ECL1–6); ECL1 is the site of several mutations associated with CF. Mutation R117H has been reported to reduce current amplitude, whereas D110H, E116K, and R117C/L/P may impair channel stability. We hypothesized that these amino acids might not be directly involved in ion conduction and permeation but may contribute to stabilizing the outer vestibule architecture in CFTR. We used cRNA injected oocytes combined with electrophysiological techniques to test this hypothesis. Mutants bearing cysteine at these sites were not functionally modified by extracellular MTS reagents and were blocked by GlyH-101 similarly to WT-CFTR. These results suggest that these three residues do not contribute directly to permeation in CFTR. In contrast, mutants D110R-, E116R-, and R117A-CFTR exhibited instability of the open state and significantly shortened burst duration compared with WT-CFTR and failed to be locked into the open state by AMP-PNP (adenosine 5′-(β,γ-imido) triphosphate); charge-retaining mutants showed mainly the full open state with comparably longer open burst duration. These interactions suggest that these ECL1 residues might be involved in maintaining the outer pore architecture of CFTR. A CFTR homology model suggested that E116 interacts with R104 in both the closed and open states, D110 interacts with K892 in the fully closed state, and R117 interacts with E1126 in the open state. These interactions were confirmed experimentally. The results suggest that D110, E116, and R117 may contribute to stabilizing the architecture of the outer pore of CFTR by interactions with other charged residues.

Copyright information:

© 2014 Cui et al.

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