Skip to main content

Electrophysiological Approach to Studying CFTR

  • Protocol
Cystic Fibrosis Methods and Protocols

Part of the book series: Methods in Molecular Medicine™ ((MIMM,volume 70))

  • 929 Accesses

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR) is aphosphorylation and ATP-dependent Cl∼ channel. It is predominantly expressed in the apical membrane of epithelial cells. The presence and function of CFTR in a cell is sensitively measured using electrophysiological techniques. The patch clamp technique is the most frequently used method to measure CFTR in single cells or in isolated patches of cell membrane. A key step for the successful recording of CFTR is its identification and distinction from other Cl∼ channels. In patch clamp recordings this is done by probing for physiological, pharmacological, or biophysical characteristics of the channel. This chapter describes first, how to successfully record CFTR currents using the patch clamp technique. Second, it focuses on current noise analysis as a useful tool to investigate the regulation of CFTR activity.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Penner, R. (1995) Pracical guide to patch clamping, in Single Channel Recording (Sakmann, B. and Neher, E., eds.), Plenum Press, New York, London, pp. 3–30.

    Google Scholar 

  2. Haws, C., Finkbeiner, W. E., Widdicombe, J. H., and Wine, J. J. (1994) CFTR in Calu-3 human airway cells: channel properties and role in cAMP-activated Cl conductance. Am. J. Physiol. Cell Physiol. 266 (10) L502–L512.

    CAS  Google Scholar 

  3. Fischer, H. and Machen, T. E. (1996) The tyrosine kinase p60c-src regulates the fast gate of the cystic fibrosis transmembrane conductance regulator chloride channel. Biophys. J. 71, 3073–3082.

    Article  CAS  PubMed  Google Scholar 

  4. Zhu, T., Dahan, D., Evagelidis, A., Zheng, S., Luo, J., and Hanrahan, J. W. (1999) Association of cystic fibrosis transmembrane conductance regulator and protein phosphatase 2C. J Biol. Chem. 274, 29,102–29,107.

    Article  CAS  PubMed  Google Scholar 

  5. Becq, F., Jensen, T. J., Chang, X. B., Savoia, A., Rommens, J. M., Tsui, L. C., Buchwald, M., Riordan, J. R., and Hanrahan, J. W. (1994) Phosphatase inhibitors activate normal and defective CFTR chloride channels. Proc. Natl. Acad. Sci. USA 91, 9160–9164.

    Article  CAS  PubMed  Google Scholar 

  6. Hwang, T. C., Horie, M., and Gadsby, D. C. (1993) Functionally distinct phosphoforms underlie incremental activation of protein kinase-regulated Cl conductance in mammalian heart. J Gen. Physiol. 101, 629–651.

    Article  CAS  PubMed  Google Scholar 

  7. Anderson, M. P. and Welsh, M. J. (1992) Regulation by ATP and ADP of CFTR chloride channels that contain mutant nucleotide-binding domains. Science 257, 1701–1704.

    Article  CAS  PubMed  Google Scholar 

  8. Julien, M., Verrier, B., Cerutti, M., Chappe, V., Gola, M., Devauchelle, G., and Becq, F. (1999) Cystic fibrosis transmembrane conductance regulator (CFTR) confers glibenclamide sensitivity to outwardly rectifying chloride channel (ORCC) in Hi-5 insect cells. J Membr. Biol. 168, 229–239.

    Article  CAS  PubMed  Google Scholar 

  9. Rabe, A., Disser, J., and Frömter, E. (1995) Cl- channel inhibition by glibenclamide is not specific for the CFTR-type Cl- channel. Pflugers Arch. 429, 659–662.

    Article  CAS  PubMed  Google Scholar 

  10. Linsdell, P. and Hanrahan, J. W. (1996) Disulphonic stilbene block of cystic fibrosis transmembrane conductance regulator Cl- channels expressed in a mammalian cell line and its regulation by a critical pore residue. J Physiol. (Lond.) 496, 687–693.

    CAS  Google Scholar 

  11. Linsdell, P. (2000) Inhibition of CFTR Cl channel currents by arachidonic acid. Can. J. Physiol. Pharmacol. 78, 490–499.

    Article  CAS  PubMed  Google Scholar 

  12. Linsdell, P. and Hanrahan, J. W. (1996) Flickery block of single CFTR chloride channels by intracellular anions and osmolytes. Am. J. Physiol. Cell Physiol. 271, C628–634.

    CAS  Google Scholar 

  13. Illek, B., Fischer, H., Santos, G. F., Widdicombe, J. H., Machen, T. E., and Reenstra, W. W. (1995) cAMP-independent activation of CFTR Cl channels by the tyrosine kinase inhibitor genistein. Am. J. Physiol. Cell Physiol. 268, C886–C893.

    CAS  Google Scholar 

  14. Illek, B. and Fischer, H. (1998) Flavonoids stimulate Cl conductance of human airway epithelium in vitro and in vivo. Am. J. Physiol. Lung Cell. Mol. Physiol. 275, L902–L910.

    CAS  Google Scholar 

  15. Illek, B., Lizarzaburu, M. E., Lee, V., Nantz, M. H., Kurth, M. J., and Fischer, H. (2000) Structural determinants for activation and block of CFTR-mediated chloride currents by apigenin. Am. J. Physiol. Cell Physiol. 279, C1838–C1846.

    CAS  PubMed  Google Scholar 

  16. Wang, F., Zeltwanger, S., Yang, I. C., Nairn, A. C., and Hwang, T. C. (1998) Actions of genistein on cystic fibrosis transmembrane conductance regulator channel gating. Evidence for two binding sites with opposite effects. J Gen. Physiol. 111, 477–490.

    Article  CAS  PubMed  Google Scholar 

  17. Berger, H. A., Anderson, M. P., Gregory, R. J., Thompson, S., Howard, P. W., Maurer, R. A., Mulligan, R., Smith, A. E., and Welsh, M. J. (1991) Identification and regulation of the cystic fibrosis transmembrane conductance regulator-generated chloride channel. J Clin. Invest. 88, 1422–1431.

    Article  CAS  PubMed  Google Scholar 

  18. Tabcharani, J. A., Low, W., Elie, D., and Hanrahan, J. W. (1990) Low conductance Cl channel activated by cAMP in the epithelial cell line T84. FEBS Lett. 270, 157–163.

    Article  CAS  PubMed  Google Scholar 

  19. Fischer, H. and Machen, T. E. (1994) CFTR displays voltage dependence and two gating modes during stimulation. J Gen. Physiol. 104, 541–566.

    Article  CAS  PubMed  Google Scholar 

  20. Winter, M. C., Sheppard, D. N., Carson, M. R., and Welsh, M. J. (1994) Effect of ATP concentration on CFTR CL channels: a kinetic analysis of channel regulation. Biophys. J. 66, pp1398–1403.

    Article  CAS  PubMed  Google Scholar 

  21. Carson, M. R., Travis, S. M., and Welsh, M. J. (1995) The two nucleotide-binding domains of cystic fibrosis transmembrane conductance regulator (CFTR) have distinct functions in controlling channel activity. J Biol. Chem. 270, 1711–1717.

    Article  CAS  PubMed  Google Scholar 

  22. Gunderson, K. L. and Kopito, R. R. (1995) Conformational states of CFTR associated with channel gating: the role ATP binding and hydrolysis. Cell 82, 231–239.

    Article  CAS  PubMed  Google Scholar 

  23. Venglarik, C. J., Schultz, B. D., Frizzell, R. A., and Bridges, R. J. (1994) ATP alters current fluctuations of cystic fibrosis transmembrane conductance regulator: evidence for a three-state activation mechanism. J Gen. Physiol. 104, 123–146.

    Article  CAS  PubMed  Google Scholar 

  24. Haws, C., Krouse, M. E., Xia, Y., Gruenert, D. C., and Wine, J. J. (1992) CFTR channels in immortalized human airway cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 263, L692–707.

    CAS  Google Scholar 

  25. Fischer, H. (1997) CFTR’s fast gate is caused by an intracellular non-diffusible factor (abstr.). Ped. Pulmonol. suppl 14, 230.

    Google Scholar 

  26. Gray, M. A., Harris, A., Coleman, L., Greenwell, J. R., and Argent, B. E. (1989) Two types of chloride channel on duct cells cultured from human fetal pancreas. Am. J. Physiol. Cell Physiol. 257, C240–251.

    CAS  Google Scholar 

  27. Baukrowitz, T., Hwang, T. C., Nairn, A. C., and Gadsby, D. C. (1994) Coupling of CFTR Cl&#201C; channel gating to an ATP hydrolysis cycle. Neuron 12, 473–482.

    Article  CAS  PubMed  Google Scholar 

  28. Gadsby, D. C. and Nairn, A. C. (1999) Control of CFTR channel gating by phosphorylation and nucleotide hydrolysis. Physiol. Rev. 79, S77–S107.

    CAS  PubMed  Google Scholar 

  29. Ehrenstein, G., Lecar, H., and Nossal, R. (1970) The nature of the negative resistance in bimolecular lipid membranes containing excitability-induced material. J Gen. Physiol. 55, 119–133.

    Article  CAS  PubMed  Google Scholar 

  30. Worrell, R. T., Butt, A. G., Cliff, W. H., and Frizzell, R. A. (1989) A volume-sensitive choride conductance in human colonic cell line T84. Am. J. Physiol. Cell Physiol. 256, C1111–C1119.

    CAS  Google Scholar 

  31. Sigworth, F. J. (1980) The variance of sodium current fluctuations at the node of ranvier. J Physiol. (Lond.) 307, 97–129.

    CAS  Google Scholar 

  32. Colquhoun, D. and Hawkes, A. G. (1995) The principles of the stochastic interpretation of ion-channel mechanisms, in Single-Channel Recording, 2nd ed. (Sakmann, B. and Neher, E., eds.), Plenum Press, New York.

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2002 Humana Press Inc.

About this protocol

Cite this protocol

Fischer, H. (2002). Electrophysiological Approach to Studying CFTR. In: Skach, W.R. (eds) Cystic Fibrosis Methods and Protocols. Methods in Molecular Medicine™, vol 70. Humana Press. https://doi.org/10.1385/1-59259-187-6:49

Download citation

  • DOI: https://doi.org/10.1385/1-59259-187-6:49

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-897-4

  • Online ISBN: 978-1-59259-187-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics