Pflügers Archiv - European Journal of Physiology

, Volume 463, Issue 3, pp 405–418

Differential contribution of TM6 and TM12 to the pore of CFTR identified by three sulfonylurea-based blockers

  • Guiying Cui
  • Binlin Song
  • Hussein W. Turki
  • Nael A. McCarty
Ion Channels, Receptors and Transporters

Abstract

Previous studies suggested that four transmembrane domains 5, 6, 11, 12 make the greatest contribution to forming the pore of the CFTR chloride channel. We used excised, inside-out patches from oocytes expressing CFTR with alanine-scanning mutagenesis in amino acids in TM6 and TM12 to probe CFTR pore structure with four blockers: glibenclamide (Glyb), glipizide (Glip), tolbutamide (Tolb), and Meglitinide. Glyb and Glip blocked wildtype (WT)-CFTR in a voltage-, time-, and concentration-dependent manner. At VM = −120 mV with symmetrical 150 mM Cl solution, fractional block of WT-CFTR by 50 μM Glyb and 200 μM Glip was 0.64 ± 0.03 (n = 7) and 0.48 ± 0.02 (n = 7), respectively. The major effects on block by Glyb and Glip were found with mutations at F337, S341, I344, M348, and V350 of TM6. Under similar conditions, fractional block of WT-CFTR by 300 μM Tolb was 0.40 ± 0.04. Unlike Glyb, Glip, and Meglitinide, block by Tolb lacked time-dependence (n = 7). We then tested the effects of alanine mutations in TM12 on block by Glyb and Glip; the major effects were found at N1138, T1142, V1147, N1148, S1149, S1150, I1151, and D1152. From these experiments, we infer that amino acids F337, S341, I344, M348, and V350 of TM6 face the pore when the channel is in the open state, while the amino acids of TM12 make less important contributions to pore function. These data also suggest that the region between F337 and S341 forms the narrow part of the CFTR pore.

Keywords

Cystic fibrosis transmembrane conductance regulator Chloride channel Sulfonylurea Channel blockade 

Supplementary material

424_2011_1035_MOESM1_ESM.doc (2 mb)
ESM doc(DOC 2.04 MB)

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Guiying Cui
    • 1
  • Binlin Song
    • 1
  • Hussein W. Turki
    • 1
  • Nael A. McCarty
    • 1
  1. 1.Division of Pulmonology, Allergy/Immunology, Cystic Fibrosis, and Sleep, Department of Pediatrics, Center for Cystic Fibrosis Research, Children’s Healthcare of AtlantaEmory University School of MedicineAtlantaUSA

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