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Contribution of the eighth transmembrane segment to the function of the CFTR chloride channel pore

  • Alexander Negoda
  • Mairin S. Hogan
  • Elizabeth A. Cowley
  • Paul LinsdellEmail author
Original Article

Abstract

Our molecular understanding of the cystic fibrosis transmembrane conductance regulator (CFTR)—the chloride channel that is mutated in cystic fibrosis—has been greatly enhanced by a number of recent atomic-level structures of the protein in different conformations. One surprising aspect of these structures was the finding that the eighth of CFTR’s 12 membrane-spanning segments (TM8) appeared close to the channel pore. Although functional evidence supports a role for other TMs in forming the pore, such a role for TM8 has not previously been reported. Here, we use patch-clamp recording to investigate the functional role of TM8. Using substituted cysteine accessibility mutagenesis, we find that three amino acid side-chains in TM8 (Y913, Y914, and Y917) are exposed to the extracellular, but not the intracellular, solution. Cysteine cross-linking experiments suggest that Y914 and Y917 are in close proximity to L102 (TM1) and F337 (TM6), respectively, suggesting that TM8 contributes to the narrow selectivity filter region of the pore. Different amino acid substitutions suggest that Y914, and to a lesser extent Y917, play important roles in controlling anion flux through the open channel. Furthermore, substitutions that reduce side-chain volume at Y917 severely affect channel gating, resulting in a channel with an extremely unstable open state. Our results suggest that pore-lining TM8 is among the most important TMs controlling the permeation phenotype of the CFTR channel, and also that movement of TM8 may be critically involved in channel gating.

Keywords

Cystic fibrosis transmembrane conductance regulator Chloride channel Substituted cysteine accessibility mutagenesis Selectivity filter Channel gating Channel structure 

Abbreviations

BHK

Baby hamster kidney

CF

Cystic fibrosis

CFTR

CF transmembrane conductance regulator

CHO

Chinese hamster ovary

Cryo-EM

Electron cryo-microscopy

CuPhe

Copper(II)-o-phenanthroline

DTT

Dithiothreitol

MSD

Membrane-spanning domain

MTS

Methanethiosulfonate

MTSES

[2-Sulfonatoethyl] MTS

MTSET

[2-(Trimethylammonium)ethyl] MTS

NBD

Nucleotide-binding domain

PKA

Protein kinase A

SCAM

Substituted cysteine accessibility mutagenesis

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.
    Wang Y, Wrennall JA, Cai Z, Li H, Sheppard DN (2014) Understanding how cystic fibrosis mutations disrupt CFTR function: from single molecules to animal models. Int J Biochem Cell Biol 52:47–57CrossRefGoogle Scholar
  2. 2.
    Csanády L, Vergani P, Gadsby DC (2019) Structure, gating, and regulation of the CFTR anion channel. Physiol Rev 99:707–738CrossRefGoogle Scholar
  3. 3.
    Moran O (2017) The gating of the CFTR channel. Cell Mol Life Sci 74:85–92CrossRefGoogle Scholar
  4. 4.
    Hwang T-C, Yeh J-T, Zhang J, Yu Y-C, Yeh H-I, Destefano S (2018) Structural mechanisms of CFTR function and dysfunction. J Gen Physiol 150:539–570Google Scholar
  5. 5.
    Zhang Z, Chen J (2016) Atomic structure of the cystic fibrosis transmembrane conductance regulator. Cell 167:1586–1597CrossRefGoogle Scholar
  6. 6.
    Liu F, Zhang Z, Csanády L, Gadsby DC, Chen J (2017) Molecular structure of the human CFTR ion channel. Cell 169:85–95CrossRefGoogle Scholar
  7. 7.
    Zhang Z, Liu F, Chen J (2017) Conformational changes of CFTR upon phosphorylation and ATP binding. Cell 170:483–491CrossRefGoogle Scholar
  8. 8.
    Zhang Z, Liu F, Chen J (2018) Molecular structure of the ATP-bound, phosphorylated human CFTR. Proc Natl Acad Sci USA 115:12757–12762CrossRefGoogle Scholar
  9. 9.
    Callebaut I, Hoffmann B, Lehn P, Mornon J-P (2017) Molecular modelling and molecular dynamics of CFTR. Cell Mol Life Sci 74:3–22CrossRefGoogle Scholar
  10. 10.
    Linsdell P (2017) Architecture and functional properties of the CFTR channel pore. Cell Mol Life Sci 74:67–83CrossRefGoogle Scholar
  11. 11.
    Linsdell P, Evagelidis A, Hanrahan JW (2000) Molecular determinants of anion selectivity in the cystic fibrosis transmembrane conductance regulator chloride channel pore. Biophys J 78:2973–2982CrossRefGoogle Scholar
  12. 12.
    McCarty NA, Zhang Z-R (2001) Identification of a region of strong discrimination in the pore of CFTR. Am J Physiol 281:L852–L867Google Scholar
  13. 13.
    Linsdell P (2016) Anion conductance selectivity mechanism of the CFTR chloride channel. Biochim Biophys Acta 1858:740–747CrossRefGoogle Scholar
  14. 14.
    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 1859:1049–1058CrossRefGoogle Scholar
  15. 15.
    Corradi V, Vergani P, Tieleman DP (2015) Cystic fibrosis transmembrane conductance regulator (CFTR): closed and open state channel models. J Biol Chem 290:22891–22906CrossRefGoogle Scholar
  16. 16.
    Gao X, Hwang T-C (2015) Localizing a gate in CFTR. Proc Natl Acad Sci USA 112:2461–2466CrossRefGoogle Scholar
  17. 17.
    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 30:1247–1262CrossRefGoogle Scholar
  18. 18.
    Fay JF, Aleksandrov LA, Jensen T, Cui L, Kousouros JN, He L, Aleksandrov AA, Gingerich D, Riordan JR, Chen J (2018) Cryo-EM visualization of an active high open probability CFTR anion channel. Biochemistry 57:6234–6246CrossRefGoogle Scholar
  19. 19.
    Corradi V, Gu R-X, Vergani P, Tieleman DP (2018) Structure of transmembrane helix 8 and possible membrane defects in CFTR. Biophys J 114:1751–1754CrossRefGoogle Scholar
  20. 20.
    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 135:229–245CrossRefGoogle Scholar
  21. 21.
    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 25:4728–4739CrossRefGoogle Scholar
  22. 22.
    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 157:1065–1071CrossRefGoogle Scholar
  23. 23.
    Negoda A, Cowley EA, El Hiani Y, Linsdell P (2018) Conformational change of the extracellular parts of the CFTR protein during channel gating. Cell Mol Life Sci 75:3027–3038CrossRefGoogle Scholar
  24. 24.
    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 92:390–396CrossRefGoogle Scholar
  25. 25.
    Wang W, El Hiani Y, Rubaiy HN, Linsdell P (2014) Relative contribution of different transmembrane segments to the CFTR chloride channel pore. Pflügers Arch 466:477–490CrossRefGoogle Scholar
  26. 26.
    El Hiani Y, Linsdell P (2015) Functional architecture of the cytoplasmic entrance to the cystic fibrosis transmembrane conductance regulator chloride channel pore. J Biol Chem 290:15855–15865CrossRefGoogle Scholar
  27. 27.
    El Hiani Y, Negoda A, Linsdell P (2016) Cytoplasmic pathway followed by chloride ions to enter the CFTR channel pore. Cell Mol Life Sci 73:1917–1925CrossRefGoogle Scholar
  28. 28.
    Linsdell P, Hanrahan JW (1998) Adenosine triphosphate-dependent asymmetry of anion permeation in the cystic fibrosis transmembrane conductance regulator chloride channel. J Gen Physiol 111:601–614CrossRefGoogle Scholar
  29. 29.
    Smith SS, Steinle ED, Meyerhoff ME, Dawson DC (1999) Cystic fibrosis transmembrane conductance regulator. Physical basis for lyotropic anion selectivity patterns. J Gen Physiol 114:799–818CrossRefGoogle Scholar
  30. 30.
    Gong X, Burbridge SM, Cowley EA, Linsdell P (2002) Molecular determinants of Au(CN)2 binding and permeability within the cystic fibrosis transmembrane conductance regulator Cl channel pore. J Physiol 540:39–47CrossRefGoogle Scholar
  31. 31.
    Ge N, Muise CN, Gong X, Linsdell P (2004) Direct comparison of the functional roles played by different transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore. J Biol Chem 279:55283–55289CrossRefGoogle Scholar
  32. 32.
    Fatehi M, Linsdell P (2009) Novel residues lining the CFTR chloride channel pore identified by functional modification of introduced cysteines. J Membr Biol 228:151–164CrossRefGoogle Scholar
  33. 33.
    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 457:351–360CrossRefGoogle Scholar
  34. 34.
    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 138:165–178CrossRefGoogle Scholar
  35. 35.
    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 104:786–797CrossRefGoogle Scholar
  36. 36.
    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 283:4957–4966CrossRefGoogle Scholar
  37. 37.
    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 136:293–309CrossRefGoogle Scholar
  38. 38.
    El Hiani Y, Linsdell P (2010) Changes in accessibility of cytoplasmic substances to the pore associated with activation of the cystic fibrosis transmembrane conductance regulator chloride channel. J Biol Chem 285:32126–32140CrossRefGoogle Scholar
  39. 39.
    Norimatsu Y, Ivetac A, Alexander C, Kirkham J, O’Donnell N, Dawson DC, Sansom MSP (2012) Cystic fibrosis transmembrane conductance regulator: a molecular model defines the architecture of the anion conduction path and locates a “bottleneck” in the pore. Biochemistry 51:2199–2212CrossRefGoogle Scholar
  40. 40.
    Bai Y, Li M, Hwang T-C (2011) Structural basis for the channel function of a degraded ABC transporter, CFTR (ABCC7). J Gen Physiol 138:495–507CrossRefGoogle Scholar
  41. 41.
    Qian F, El Hiani Y, Linsdell P (2011) Functional arrangement of the 12th transmembrane region in the CFTR chloride channel based on functional investigation of a cysteine-less variant. Pflügers Arch 462:559–571CrossRefGoogle Scholar
  42. 42.
    Zhang J, Hwang T-C (2015) The fifth transmembrane segment of cystic fibrosis transmembrane conductance regulator contributes to its anion permeation pathway. Biochemistry 54:3839–3850CrossRefGoogle Scholar
  43. 43.
    Holstead RG, Li M-S, Linsdell P (2011) Functional differences in pore properties between wild-type and cysteine-less forms of the CFTR chloride channel. J Membr Biol 243:15–23CrossRefGoogle Scholar
  44. 44.
    Linsdell P, Zheng S-X, Hanrahan JW (1998) Non-pore lining amino acid side chains influence anion selectivity of the human CFTR Cl channel expressed in mammalian cell lines. J Physiol 512:1–16CrossRefGoogle Scholar
  45. 45.
    McDonough S, Davidson N, Lester HA, McCarty NA (1994) Novel pore-lining residues in CFTR that govern permeation and open-channel block. Neuron 13:623–634CrossRefGoogle Scholar
  46. 46.
    Gupta J, Linsdell P (2003) Extent of the selectivity filter conferred by the sixth transmembrane region in the CFTR chloride channel pore. Mol Membr Biol 20:45–52CrossRefGoogle Scholar
  47. 47.
    Linsdell P (2015) Metal bridges to probe membrane ion channel structure and function. Biomol Concepts 6:191–203CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Alexander Negoda
    • 1
  • Mairin S. Hogan
    • 1
  • Elizabeth A. Cowley
    • 1
  • Paul Linsdell
    • 1
    Email author
  1. 1.Department of Physiology and BiophysicsDalhousie UniversityHalifaxCanada

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