Mutations at Arginine 352 Alter the Pore Architecture of CFTR

  • Guiying Cui
  • Zhi-Ren Zhang
  • Andrew R. W. O’Brien
  • Binlin Song
  • Nael A. McCarty
Article

Abstract

Arginine 352 (R352) in the sixth transmembrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR) previously was reported to form an anion/cation selectivity filter and to provide positive charge in the intracellular vestibule. However, mutations at this site have nonspecific effects, such as inducing susceptibility of endogenous cysteines to chemical modification. We hypothesized that R352 stabilizes channel structure and that charge-destroying mutations at this site disrupt pore architecture, with multiple consequences. We tested the effects of mutations at R352 on conductance, anion selectivity and block by the sulfonylurea drug glipizide, using recordings of wild-type and mutant channels. Charge-altering mutations at R352 destabilized the open state and altered both selectivity and block. In contrast, R352K-CFTR was similar to wild-type. Full conductance state amplitude was similar to that of wild-type CFTR in all mutants except R352E, suggesting that R352 does not itself form an anion coordination site. In an attempt to identify an acidic residue that may interact with R352, we found that permeation properties were similarly affected by charge-reversing mutations at D993. Wild-type-like properties were rescued in R352E/D993R-CFTR, suggesting that R352 and D993 in the wild-type channel may interact to stabilize pore architecture. Finally, R352A-CFTR was sensitive to modification by externally applied MTSEA+, while wild-type and R352E/D993R-CFTR were not. These data suggest that R352 plays an important structural role in CFTR, perhaps reflecting its involvement in forming a salt bridge with residue D993.

Keywords

CFTR Chloride channel Salt bridge Channel open state Subconductance Blocker Anion selectivity 

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Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Guiying Cui
    • 1
    • 2
  • Zhi-Ren Zhang
    • 1
  • Andrew R. W. O’Brien
    • 1
  • Binlin Song
    • 1
  • Nael A. McCarty
    • 1
    • 2
  1. 1.School of BiologyGeorgia Institute of TechnologyAtlantaUSA
  2. 2.Division of Pulmonology, Allergy/Immunology, Cystic Fibrosis, and Sleep, Department of PediatricsEmory University School of MedicineAtlantaUSA

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