Skip to main content
SpringerLink
Log in

Application of High-Resolution Single-Channel Recording to Functional Studies of Cystic Fibrosis Mutants

  • Protocol
  • First Online:
Book cover Cystic Fibrosis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 741))

Abstract

The patch-clamp technique is a powerful and versatile method to investigate the cystic fibrosis transmembrane conductance regulator (CFTR) Cl– channel, its malfunction in disease and modulation by small molecules. Here, we discuss how the molecular behaviour of CFTR is investigated using high-resolution single-channel recording and kinetic analyses of channel gating. We review methods used to quantify how cystic fibrosis (CF) mutants perturb the biophysical properties and regulation of CFTR. By explaining the relationship between macroscopic and single-channel currents, we demonstrate how single-channel data provide molecular explanations for changes in CFTR-mediated transepithelial ion transport elicited by CF mutants.

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

Access this chapter

Log in via an institution
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. Neher, E., and Sakmann, B. (1976) Single-channel currents recorded from membrane of denervated frog muscle fibres. Nature 260, 799–802.

    Article  PubMed  CAS  Google Scholar 

  2. Hamill, O. P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F. J. (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 391, 85–100.

    Article  PubMed  CAS  Google Scholar 

  3. Ostedgaard, L. S., Baldursson, O., and Welsh, M. J. (2001) Regulation of the cystic fibrosis transmembrane conductance regulator Cl– channel by its R domain. J. Biol. Chem. 276, 7689–7692.

    Article  PubMed  CAS  Google Scholar 

  4. Gadsby, D. C., Vergani, P., and Csanády, L. (2006) The ABC protein turned chloride channel whose failure causes cystic fibrosis. Nature 440, 477–483.

    Article  PubMed  CAS  Google Scholar 

  5. Hwang, T. C., and Sheppard, D. N. (2009) Gating of the CFTR Cl– channel by ATP-driven nucleotide-binding domain dimerisation. J. Physiol. 587, 2151–2161.

    Article  PubMed  CAS  Google Scholar 

  6. Sakmann, B., and Neher, E. (1995) Single-channel recording, 2nd ed. Plenum, New York, NY.

    Google Scholar 

  7. Hug, M. J. (2002) The whole-cell patch-clamp technique – a powerful tool to approach CFTR function. Virtual Repository of Methods and Reagents for CFTR Expression and Functional Studies. http://central.igc.gulbenkian.pt/cftr/vr/physiology.html. Accessed 11 March 2010.

  8. Gray, M. A. (2002) Investigating the properties of the CFTR channel using the cell-attached configuration of the patch clamp – monitoring single channel activity in a living cell. Virtual Repository of Methods and Reagents for CFTR Expression and Functional Studies. http://central.igc.gulbenkian.pt/cftr/vr/physiology.html. Accessed 11 March 2010.

  9. Hanrahan, J. W., Kone, Z., Mathews, C. J., Luo, J., Jia, Y., and Linsdell, P. (1998) Patch-clamp studies of cystic fibrosis transmembrane conductance regulator chloride channel. Methods Enzymol. 293, 169–194.

    Article  PubMed  CAS  Google Scholar 

  10. Scott-Ward, T. S., Chen, J. H., Li, H., Cai, Z., and Sheppard, D. N. (2002) Measurement of Cl– flow through CFTR channels using the excised inside-out patch-clamp Virtual Repository of Methods and Reagents for CFTR Expression and Functional Studies. http://central.igc.gulbenkian.pt/cftr/vr/physiology.html. Accessed 11 March 2010.

  11. Gong, X., Gupta, J., and Linsdell, P. (2002) Measurement of the permeation and conduction properties of the CFTR chloride channel using excised inside-out membrane. Virtual Repository of Methods and Reagents for CFTR Expression and Functional Studies. http://central.igc.gulbenkian.pt/cftr/vr/physiology.html. Accessed 11 March 2010.

  12. Sohma, Y., and Hwang, T. C. (2002) Kinetic analysis of CFTR single-channel data. Virtual Repository of Methods and Reagents for CFTR Expression and Functional Studies. http://central.igc.gulbenkian.pt/cftr/vr/physiology.html. Accessed 11 March 2010.

  13. Cai, Z., Scott-Ward, T. S., Li, H., Schmidt, A., and Sheppard, D. N. (2002) Strategies to investigate the mechanism of action of CFTR modulators Virtual Repository of Methods and Reagents for CFTR Expression and Functional Studies. http://central.igc.gulbenkian.pt/cftr/vr/physiology.html. Accessed 11 March 2010.

  14. Sheppard, D. N., and Robinson, K. A. (1997) Mechanism of glibenclamide inhibition of cystic fibrosis transmembrane conductance regulator Cl– channels expressed in a murine cell line. J. Physiol. 503, 333–346.

    Article  PubMed  CAS  Google Scholar 

  15. Lansdell, K. A., Delaney, S. J., Lunn, D. P., Thomson, S. A., Sheppard, D. N., and Wainwright, B. J. (1998) Comparison of the gating behaviour of human and murine cystic fibrosis transmembrane conductance regulator Cl– channels expressed in mammalian cells. J. Physiol. 508, 379–392.

    Article  PubMed  CAS  Google Scholar 

  16. Anderson, M. P., Gregory, R. J., Thompson, S., Souza, D. W., Paul, S., Mulligan, R. C., et al. (1991) Demonstration that CFTR is a chloride channel by alteration of its anion selectivity. Science 253, 202–205.

    Article  PubMed  CAS  Google Scholar 

  17. Cheng, S. H., Gregory, R. J., Marshall, J., Paul, S., Souza, D. W., White, G. A., et al. (1990) Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell 63, 827–834.

    Article  PubMed  CAS  Google Scholar 

  18. Drumm, M. L., Wilkinson, D. J., Smit, L. S., Worrell, R. T., Strong, T. V., Frizzell, R. A., et al. (1991) Chloride conductance expressed by ΔF508 and other mutant CFTRs in Xenopus oocytes. Science 254, 1797–1799.

    Article  PubMed  CAS  Google Scholar 

  19. Scott-Ward, T. S., Cai, Z., Dawson, E. S., Doherty, A., Da Paula, A. C., Davidson, H., et al. (2007) Chimeric constructs endow the human CFTR Cl– channel with the gating behavior of murine CFTR. Proc. Natl. Acad. Sci. USA 104, 16365–16370.

    Article  PubMed  CAS  Google Scholar 

  20. Wu, J. V., Joo, N. S., Krouse, M. E., and Wine, J. J. (2001) Cystic fibrosis transmembrane conductance regulator gating requires cytosolic electrolytes. J. Biol. Chem. 276, 6473–6478.

    Article  PubMed  CAS  Google Scholar 

  21. Ishihara, H., and Welsh, M. J. (1997) Block by MOPS reveals a conformation change in the CFTR pore produced by ATP hydrolysis. Am. J. Physiol. 273, C1278–C1289.

    PubMed  CAS  Google Scholar 

  22. Tabcharani, J. A., Chang, X. B., Riordan, J. R., and Hanrahan, J. W. (1991) Phosphorylation-regulated Cl– channel in CHO cells stably expressing the cystic fibrosis gene. Nature 352, 628–631.

    Article  PubMed  CAS  Google Scholar 

  23. Chen, J. H., Cai, Z., and Sheppard, D. N. (2009) Direct sensing of intracellular pH by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl– channel. J. Biol. Chem. 284, 35495–35506.

    Article  PubMed  CAS  Google Scholar 

  24. Hughes, L. K., Ju, M., and Sheppard, D. N. (2008) Potentiation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl– currents by the chemical solvent tetrahydrofuran. Mol. Membr. Biol. 25, 528–538.

    Article  PubMed  CAS  Google Scholar 

  25. Cai, Z., Scott-Ward, T. S., and Sheppard, D. N. (2003) Voltage-dependent gating of the cystic fibrosis transmembrane conductance regulator Cl– channel. J. Gen. Physiol. 122, 605–620.

    Article  PubMed  CAS  Google Scholar 

  26. Zhou, Z., Hu, S., and Hwang, T. C. (2001) Voltage-dependent flickery block of an open cystic fibrosis transmembrane conductance regulator (CFTR) channel pore. J. Physiol. 532, 435–448.

    Article  PubMed  CAS  Google Scholar 

  27. Anderson, M. P., Berger, H. A., Rich, D. P., Gregory, R. J., Smith, A. E., and Welsh, M. J. (1991) Nucleoside triphosphates are required to open the CFTR chloride channel. Cell 67, 775–784.

    Article  PubMed  CAS  Google Scholar 

  28. Schultz, B. D., Frizzell, R. A., and Bridges, R. J. (1999) Rescue of dysfunctional ΔF508-CFTR chloride channel activity by IBMX. J. Membr. Biol. 170, 51–66.

    Article  PubMed  CAS  Google Scholar 

  29. Bompadre, S. G., Cho, J. H., Wang, X., Zou, X., Sohma, Y., Li, M., et al. (2005) CFTR gating II: effects of nucleotide binding on the stability of open states. J. Gen. Physiol. 125, 377–394.

    Article  PubMed  CAS  Google Scholar 

  30. 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. 496, 687–693.

    PubMed  CAS  Google Scholar 

  31. Zhou, Z., Hu, S., and Hwang, T. C. (2002) Probing an open CFTR pore with organic anion blockers. J. Gen. Physiol. 120, 647–662.

    Article  PubMed  CAS  Google Scholar 

  32. Csanády, L., Chan, K. W., Seto-Young, D., Kopsco, D. C., Nairn, A. C., and Gadsby, D. C. (2000) Severed channels probe regulation of gating of cystic fibrosis transmembrane conductance regulator by its cytoplasmic domains. J. Gen. Physiol. 116, 477–500.

    Article  PubMed  Google Scholar 

  33. Cai, Z., and Sheppard, D. N. (2002) Phloxine B interacts with the cystic fibrosis transmembrane conductance regulator at multiple sites to modulate channel activity. J. Biol. Chem. 277, 19546–19553.

    Article  PubMed  CAS  Google Scholar 

  34. Hwang, T. C., Nagel, G., Nairn, A. C., and Gadsby, D. C. (1994) Regulation of the gating of cystic fibrosis transmembrane conductance regulator C1 channels by phosphorylation and ATP hydrolysis. Proc. Natl. Acad. Sci. USA 91, 4698–4702.

    Article  PubMed  CAS  Google Scholar 

  35. Carson, M. R., Winter, M. C., Travis, S. M., and Welsh, M. J. (1995) Pyrophosphate stimulates wild-type and mutant cystic fibrosis transmembrane conductance regulator Cl– channels. J. Biol. Chem. 270, 20466–20472.

    Article  PubMed  CAS  Google Scholar 

  36. Tsai, M. F., Shimizu, H., Sohma, Y., Li, M., and Hwang, T. C. (2009) State-dependent modulation of CFTR gating by pyrophosphate. J. Gen. Physiol. 133, 405–419.

    Article  PubMed  CAS  Google Scholar 

  37. Cai, Z., Taddei, A., and Sheppard, D. N. (2006) Differential sensitivity of the cystic fibrosis (CF)-associated mutants G551D and G1349D to potentiators of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl– channel. J. Biol. Chem. 281, 1970–1977.

    Article  PubMed  CAS  Google Scholar 

  38. Bompadre, S. G., Sohma, Y., Li, M., and Hwang, T. C. (2007) G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects. J. Gen. Physiol. 129, 285–298.

    Article  PubMed  CAS  Google Scholar 

  39. 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  PubMed  CAS  Google Scholar 

  40. Zeltwanger, S., Wang, F., Wang, G. T., Gillis, K. D., and Hwang, T. C. (1999) Gating of cystic fibrosis transmembrane conductance regulator chloride channels by adenosine triphosphate hydrolysis. Quantitative analysis of a cyclic gating scheme. J. Gen. Physiol. 113, 541–554.

    Article  PubMed  CAS  Google Scholar 

  41. Aleksandrov, A. A., and Riordan, J. R. (1998) Regulation of CFTR ion channel gating by MgATP. FEBS Lett. 431, 97–101.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  43. Bompadre, S. G., Ai, T., Cho, J. H., Wang, X., Sohma, Y., Li, M., et al. (2005) CFTR gating I: characterization of the ATP-dependent gating of a phosphorylation-independent CFTR channel (ΔR-CFTR). J. Gen. Physiol. 125, 361–375.

    Article  PubMed  CAS  Google Scholar 

  44. Sigurdson, W. J., Morris, C. E., Brezden, B. L., and Gardner, D. R. (1987) Stretch activation of a K+ channel in molluscan heart cells. J. Exp. Biol. 127, 191–209.

    Google Scholar 

  45. Li, C., Ramjeesingh, M., Wang, W., Garami, E., Hewryk, M., Lee, D., et al. (1996) ATPase activity of the cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 271, 28463–28468.

    Article  PubMed  CAS  Google Scholar 

  46. Weinreich, F., Riordan, J. R., and Nagel, G. (1999) Dual effects of ADP and adenylylimidodiphosphate on CFTR channel kinetics show binding to two different nucleotide binding sites. J. Gen. Physiol. 114, 55–70.

    Article  PubMed  CAS  Google Scholar 

  47. Vergani, P., Lockless, S. W., Nairn, A. C., and Gadsby, D. C. (2005) CFTR channel opening by ATP-driven tight dimerization of its nucleotide-binding domains. Nature 433, 876–880.

    Article  PubMed  CAS  Google Scholar 

  48. Vergani, P., Nairn, A. C., and Gadsby, D. C. (2003) On the mechanism of MgATP-dependent gating of CFTR Cl– channels. J. Gen. Physiol. 121, 17–36.

    Article  PubMed  CAS  Google Scholar 

  49. Zhou, Z., Wang, X., Liu, H. Y., Zou, X., Li, M., and Hwang, T. C. (2006) The two ATP binding sites of cystic fibrosis transmembrane conductance regulator (CFTR) play distinct roles in gating kinetics and energetics. J. Gen. Physiol. 128, 413–422.

    Article  PubMed  CAS  Google Scholar 

  50. Csanády, L., Vergani, P., and Gadsby, D. C. (2010) Strict coupling between CFTR’s catalytic cycle and gating of its Cl– ion pore revealed by distributions of open channel burst durations. Proc. Natl. Acad. Sci. USA 107, 1241–1246.

    Article  PubMed  Google Scholar 

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

    Google Scholar 

  52. Blatz, A. L., and Magleby, K. L. (1986) Quantitative description of three modes of activity of fast chloride channels from rat skeletal muscle. J. Physiol. 378, 141–174.

    PubMed  CAS  Google Scholar 

  53. Welsh, M. J., and Smith, A. E. (1993) Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis. Cell 73, 1251–1254.

    Article  PubMed  CAS  Google Scholar 

  54. Zielenski, J., and Tsui, L. C. (1995) Cystic fibrosis: genotypic and phenotypic variations. Annu. Rev. Genet. 29, 777–807.

    Article  PubMed  CAS  Google Scholar 

  55. Hwang, T. C., Wang, F., Yang, I. C., and Reenstra, W. W. (1997) Genistein potentiates wild-type and F508-CFTR channel activity. Am. J. Physiol. 273, C988–C998.

    PubMed  CAS  Google Scholar 

  56. Al-Nakkash, L., and Hwang, T. C. (1999) Activation of wild-type and ΔF508-CFTR by phosphodiesterase inhibitors through cAMP-dependent and -independent mechanisms. Pflugers Arch. 437, 553–561.

    Article  PubMed  CAS  Google Scholar 

  57. Dalemans, W., Barbry, P., Champigny, G., Jallat, S., Dott, K., Dreyer, D., et al. (1991) Altered chloride ion channel kinetics associated with the ΔF508 cystic fibrosis mutation. Nature 354, 526–528.

    Article  PubMed  CAS  Google Scholar 

  58. Hegedus, T., Aleksandrov, A., Cui, L., Gentzsch, M., Chang, X. B., and Riordan, J. R. (2006) F508del CFTR with two altered RXR motifs escapes from ER quality control but its channel activity is thermally sensitive. Biochim. Biophys. Acta 1758, 565–572.

    Article  PubMed  CAS  Google Scholar 

  59. Sheppard, D. N., Rich, D. P., Ostedgaard, L. S., Gregory, R. J., Smith, A. E., and Welsh, M. J. (1993) Mutations in CFTR associated with mild-disease-form Cl– channels with altered pore properties. Nature 362, 160–164.

    Article  PubMed  CAS  Google Scholar 

  60. Gong, X., and Linsdell, P. (2004) Maximization of the rate of chloride conduction in the CFTR channel pore by ion-ion interactions. Arch. Biochem. Biophys. 426, 78–82.

    Article  PubMed  CAS  Google Scholar 

  61. Linsdell, P. (2001) Relationship between anion binding and anion permeability revealed by mutagenesis within the cystic fibrosis transmembrane conductance regulator chloride channel pore. J. Physiol. 531, 51–66.

    Article  PubMed  CAS  Google Scholar 

  62. Sheppard, D. N., Travis, S. M., Ishihara, H., and Welsh, M. J. (1996) Contribution of proline residues in the membrane-spanning domains of cystic fibrosis transmembrane conductance regulator to chloride channel function. J. Biol. Chem. 271, 14995–15001.

    Article  PubMed  CAS  Google Scholar 

  63. Akabas, M. H., Kaufmann, C., Cook, T. A., and Archdeacon, P. (1994) Amino acid residues lining the chloride channel of the cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 269, 14865–14868.

    PubMed  CAS  Google Scholar 

  64. Zegarra-Moran, O., Romio, L., Folli, C., Caci, E., Becq, F., Vierfond, J. M., et al. (2002) Correction of G551D-CFTR transport defect in epithelial monolayers by genistein but not by CPX or MPB-07. Br. J. Pharmacol. 137, 504–512.

    Article  PubMed  CAS  Google Scholar 

  65. Sheppard, D. N., Ostedgaard, L. S., Winter, M. C., and Welsh, M. J. (1995) Mechanism of dysfunction of two nucleotide binding domain mutations in cystic fibrosis transmembrane conductance regulator that are associated with pancreatic sufficiency. EMBO J. 14, 876–883.

    PubMed  CAS  Google Scholar 

  66. Haws, C. M., Nepomuceno, I. B., Krouse, M. E., Wakelee, H., Law, T., Xia, Y., et al. (1996) ΔF508-CFTR channels: kinetics, activation by forskolin, and potentiation by xanthines. Am. J. Physiol. 270, C1544–C1555.

    PubMed  CAS  Google Scholar 

  67. Wang, F., Zeltwanger, S., Hu, S., and Hwang, T. C. (2000) Deletion of phenylalanine 508 causes attenuated phosphorylation-dependent activation of CFTR chloride channels. J. Physiol. 524, 637–648.

    Article  PubMed  CAS  Google Scholar 

  68. Sheppard, D. N., and Ostedgaard, L. S. (1996) Understanding how cystic fibrosis mutations cause a loss of Cl– channel function. Mol. Med. Today 2, 290–297.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank our laboratory colleagues for valuable discussions. During the preparation of this chapter, DN Sheppard was supported by the Cystic Fibrosis Trust and EuroCareCF (LSHM-CT-2005-018932), Y Sohma by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (JSPS) (19590215 and 22590212) and T-C Hwang by the National Institutes of Health (R01DK55835 and R01HL53445) and Cystic Fibrosis Foundation.

Author information

Authors and Affiliations

  1. Department of Physiology and Pharmacology, School of Medical Sciences, University of Bristol, Bristol, UK

    Zhiwei Cai & David N. Sheppard

  2. Department of Pharmacology and Neuroscience, Keio University School of Medicine, Tokyo, Japan

    Yoshiro Sohma

  3. Department of Medical Pharmacology and Physiology, Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO, USA

    Silvia G. Bompadre & Tzyh-Chang Hwang

Authors
  1. Zhiwei Cai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yoshiro Sohma
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Silvia G. Bompadre
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. David N. Sheppard
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Tzyh-Chang Hwang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Zhiwei Cai .

Editor information

Editors and Affiliations

  1. Faculty of Sciences, Centre for Biodiversity & Functional, University of Lisboa, Campo Grande, Lisboa, 1749-016, Portugal

    Margarida D. Amaral

  2. , Department of Physiology, University of Regensburg, Universitätsstr. 31, Regensburg, 93053, Germany

    Karl Kunzelmann

Rights and permissions

Reprints and permissions

Copyright information

© 2011 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Cai, Z., Sohma, Y., Bompadre, S.G., Sheppard, D.N., Hwang, TC. (2011). Application of High-Resolution Single-Channel Recording to Functional Studies of Cystic Fibrosis Mutants. In: Amaral, M., Kunzelmann, K. (eds) Cystic Fibrosis. Methods in Molecular Biology, vol 741. Humana Press. https://doi.org/10.1007/978-1-61779-117-8_27

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-117-8_27

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-116-1

  • Online ISBN: 978-1-61779-117-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation