Conformational change of the extracellular parts of the CFTR protein during channel gating

  • Alexander Negoda
  • Elizabeth A. Cowley
  • Yassine El Hiani
  • Paul Linsdell
Original Article
  • 62 Downloads

Abstract

Cystic fibrosis can be treated by potentiators, drugs that interact directly with the cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel to increase its open probability. These substances likely target key conformational changes occurring during channel opening and closing, however, the molecular bases of these conformational changes, and their susceptibility to manipulation are poorly understood. We have used patch clamp recording to identify changes in the three-dimensional organization of the extracellularly accessible parts of the CFTR protein during channel opening and closing. State-dependent formation of both disulfide bonds and Cd2+ bridges occurred for pairs of cysteine side-chains introduced into the extreme extracellular ends of transmembrane helices (TMs) 1, 6, and 12. Between each of these three TMs, we found that both disulfide bonds and metal bridges formed preferentially or exclusively in the closed state and that these inter-TM cross-links stabilized the closed state. These results indicate that the extracellular ends of these TMs are close together when the channel is closed and that they separate from each other when the channel opens. These findings identify for the first time key conformational changes in the extracellular parts of the CFTR protein that can potentially be manipulated to control channel activity.

Keywords

Cystic fibrosis transmembrane conductance regulator Chloride channel Cysteine cross-linking Conformational change Potentiator Channel structure 

Abbreviations

ABC

ATP-binding cassette

CF

Cystic fibrosis

CFTR

CF transmembrane conductance regulator

CHO

Chinese hamster ovary

cryo-EM

Cryo-electron microscopy

CuPhe

Copper(II)-o-phenanthroline

DTT

Dithiothreitol

ECL

Extracellular loop

NBD

Nucleotide-binding domain

TM

Transmembrane helix

Notes

Acknowledgements

We would like to thank Christina Irving for technical assistance. This work was supported by Cystic Fibrosis Canada.

References

  1. 1.
    Bosch B, De Boeck K (2016) Searching for a cure for cystic fibrosis. A 25-year quest in a nutshell. Eur J Pediatr 175:1–8CrossRefPubMedGoogle Scholar
  2. 2.
    Zegarra-Moran O, Galieta LJV (2017) CFTR pharmacology. Cell Mol Life Sci 74:117–128CrossRefPubMedGoogle Scholar
  3. 3.
    Linsdell P (2017) Structural changes fundamental to gating of the cystic fibrosis transmembrane conductance regulator anion channel pore. Adv Exp Med Biol 925:13–32CrossRefPubMedGoogle Scholar
  4. 4.
    Callebaut I, Hoffmann B, Mornon J-P (2017) The implications of CFTR structural studies for cystic fibrosis drug development. Curr Opin Pharmacol 34:112–118CrossRefPubMedGoogle Scholar
  5. 5.
    Chin S, Hung M, Bear CE (2017) Current insights into the role of PKA phosphorylation in CFTR channel activity and the pharmacological rescue of cystic fibrosis disease-causing mutants. Cell Mol Life Sci 74:57–66CrossRefPubMedGoogle Scholar
  6. 6.
    Moran O (2017) The gating of the CFTR channel. Cell Mol Life Sci 74:85–92CrossRefPubMedGoogle Scholar
  7. 7.
    Sorum B, Czégé D, Csanády L (2015) Timing of CFTR pore opening and structure of its transition state. Cell 163:724–733CrossRefPubMedGoogle Scholar
  8. 8.
    Gao X, Hwang T-C (2015) Localizing a gate in CFTR. Proc Natl Acad Sci USA 112:2461–2466CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Wei S, Roessler BC, Icyuz M, Chauvet S, Tao B, Hartman JL, Kirk KL (2016) Long-range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels. FASEB J 30:1247–1262CrossRefPubMedGoogle Scholar
  10. 10.
    Liu F, Zhang Z, Csanády L, Gadsby DC, Chen J (2017) Molecular structure of the human CFTR ion channel. Cell 169:85–95CrossRefPubMedGoogle Scholar
  11. 11.
    Zhang Z, Liu F, Chen J (2017) Conformational changes of CFTR upon phosphorylation and ATP binding. Cell 170:483–491CrossRefPubMedGoogle Scholar
  12. 12.
    Tordai H, Leveles I, Hegedüs T (2017) Molecular dynamics of the cryo-EM CFTR structure. Biochem Biophys Res Commun 491:986–993CrossRefPubMedGoogle Scholar
  13. 13.
    El Hiani Y, Linsdell P (2014) Metal bridges illuminate transmembrane domain movements during gating of the cystic fibrosis transmembrane conductance regulator chloride channel. J Biol Chem 289:28149–28159CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Linsdell P (2017) Architecture and functional properties of the CFTR channel pore. Cell Mol Life Sci 74:67–83CrossRefPubMedGoogle Scholar
  15. 15.
    Zhang Z-R, Song B, McCarty NA (2005) State-dependent chemical reactivity of an engineered cysteine reveals conformational changes in the outer vestibule of the cystic fibrosis transmembrane conductance regulator. J Biol Chem 280:41997–42003CrossRefPubMedGoogle Scholar
  16. 16.
    Beck EJ, Yang Y, Yaemsiri S, Raghuram V (2008) Conformational changes in a pore-lining helix coupled to cystic fibrosis transmembrane conductance regulator channel gating. J Biol Chem 283:4957–4966CrossRefPubMedGoogle Scholar
  17. 17.
    Wang W, Linsdell P (2012) Alternating access to the transmembrane domain of the ATP-binding cassette protein cystic fibrosis transmembrane conductance regulator (ABCC7). J Biol Chem 287:10156–10165CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Wang W, Linsdell P (2012) Relative movements of transmembrane regions at the outer mouth of the cystic fibrosis transmembrane conductance regulator channel pore during channel gating. J Biol Chem 287:32136–32146CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Zhou J-J, Li M-S, Qi J, Linsdell P (2010) Regulation of conductance by the number of fixed positive charges in the intracellular vestibule of the CFTR chloride channel pore. J Gen Physiol 135:229–245CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Mense M, Vergani P, White DM, Altberg G, Nairn AC, Gadsby DC (2006) In vivo phosphorylation of CFTR promotes formation of a nucleotide-binding domain heterodimer. EMBO J 25:4728–4739CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Li M-S, Demsey AFA, Qi J, Linsdell P (2009) Cysteine-independent inhibition of the CFTR chloride channel by the cysteine-reactive reagent sodium (2-sulphonatoethyl) methanethiosulphonate. Br J Pharmacol 157:1065–1071CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Zhou J-J, Fatehi M, Linsdell P (2008) Identification of positive charges situated at the outer mouth of the CFTR chloride channel pore. Pflügers Arch 457:351–360CrossRefPubMedGoogle Scholar
  23. 23.
    Fatehi M, Linsdell P (2009) Novel residues lining the CFTR chloride channel pore identified by functional modification of introduced cysteines. J Membr Biol 228:151–164CrossRefPubMedGoogle Scholar
  24. 24.
    Gao X, Bai Y, Hwang T-C (2013) Cysteine scanning of CFTR’s first transmembrane segment reveals its plausible roles in gating and permeation. Biophys J 104:786–797CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Vergani P, Nairn AC, Gadsby DC (2003) On the mechanism of MgATP-dependent gating of CFTR Cl channels. J Gen Physiol 121:17–36CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Broadbent SD, Wang W, Linsdell P (2014) Interaction between two extracellular loops influences the activity of the cystic fibrosis transmembrane conductance regulator chloride channel. Biochem Cell Biol 92:390–396CrossRefPubMedGoogle Scholar
  27. 27.
    Negoda A, El Hiani Y, Cowley EA, Linsdell P (2017) Contribution of a leucine residue in the first transmembrane segment to the selectivity filter region in the CFTR chloride channel. Biochim Biophys Acta 1859:1049–1058CrossRefPubMedGoogle Scholar
  28. 28.
    Linsdell P (2015) Metal bridges to probe membrane ion channel structure and function. Biomol Concepts 6:191–203CrossRefPubMedGoogle Scholar
  29. 29.
    Holmgren M, Shin KS, Yellen G (1998) The activation gate of a voltage-gated K+ channel can be trapped in the open state by an inter-subunit metal bridge. Neuron 21:617–621CrossRefPubMedGoogle Scholar
  30. 30.
    Heymann G, Dai J, Li M, Silberberg SD, Zhou H-X, Swartz KJ (2013) Inter- and intrasubunit interactions between transmembrane helices in the open state of P2X receptor channels. Proc Natl Acad Sci USA 110:E4045–E4054CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Zhou Y, Xia X-M, Lingle CJ (2015) Cadmium-cysteine coordination in the BK inner pore region and its structural and functional implications. Proc Natl Acad Sci USA 112:5237–5242CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Corradi V, Vergani P, Tieleman DP (2015) Cystic fibrosis transmembrane conductance regulator (CFTR): closed and open state channel models. J Biol Chem 290:22891–22906CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Bai Y, Li M, Hwang T-C (2010) Dual roles of the sixth transmembrane segment of the CFTR chloride channel in gating and permeation. J Gen Physiol 136:293–309CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Bai Y, Li M, Hwang T-C (2011) Structural basis for the channel function of a degraded ABC transporter, CFTR (ABCC7). J Gen Physiol 138:495–507CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Wang W, El Hiani Y, Linsdell P (2011) Alignment of transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore. J Gen Physiol 138:165–178CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Gao X, Hwang T-C (2016) Spatial positioning of CFTR’s pore-lining residues affirms an asymmetrical contribution of transmembrane segments to the anion permeation pathway. J Gen Physiol 147:407–422CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  • Alexander Negoda
    • 1
  • Elizabeth A. Cowley
    • 1
  • Yassine El Hiani
    • 1
  • Paul Linsdell
    • 1
  1. 1.Department of Physiology and BiophysicsDalhousie UniversityHalifaxCanada

Personalised recommendations