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Introduction to Section V: Assessment of CFTR Function

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Cystic Fibrosis

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

Abstract

This chapter introduces the various techniques to asses the function of CFTR. The numerous functional interactions of CFTR and cellular properties affected by CFTR will be described initially. This will be followed by sections explaining the importance of patch clamping and double electrode voltage clamp experiments in Xenopus oocytes for expression analysis of CFTR, and the Ussing chamber technique to analyze CFTR in polarized epithelia. It is concluded that examining CFTR function should occur at different levels, starting with the intact epithelium and ending with isolated CFTR proteins.

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References

  1. Kunzelmann, K. (2001) CFTR: interacting with everything? News Phys. Sci. 17, 167–170.

    Google Scholar 

  2. Valverde, M. A., O’Briens, J. A., Sepulveda, F. V., Ratcliff, R. A., Evans, M. J., and Colledge, W. H. (1995) Impaired cell volume regulation in intestinal crypt epithelia of cystic fibrosis. Proc. Natl. Acad. Sci. USA 92, 9038–9041.

    Article  PubMed  CAS  Google Scholar 

  3. Arniges, M., Vazquez, E., Fernandez-Fernandez, J. M., and Valverde, M. A. (2004) Swelling-activated Ca2+ entry via TRPV4 channel is defective in cystic fibrosis airway epithelia. J. Biol. Chem. 279, 54062–54068.

    Article  PubMed  CAS  Google Scholar 

  4. Kunzelmann, K., Pavenstädt, H., and Greger, R. (1989) Properties and regulation of chloride channels in cystic fibrosis and normal airway epithelial cells. Pflügers Arch. 415, 172–182.

    Article  PubMed  CAS  Google Scholar 

  5. Gabriel, S. E., Clarke, L. L., Boucher, R. C., and Stutts, M. J. (1993) CFTR and outward rectifying chloride channels are distinct proteins with a regulatory relationship. Nature 363, 263–268.

    Article  PubMed  CAS  Google Scholar 

  6. Bertrand, C. A., Zhang, R., Pilewski, J. M., and Frizzell, R. A. (2009) SLC26A9 is a constitutively active, CFTR-regulated anion conductance in human bronchial epithelia. J. Gen. Physiol. 133, 421–438.

    Article  PubMed  CAS  Google Scholar 

  7. Chang, M. H., Plata, C., Sindic, A., Ranatunga, W. K., Chen, A. P., Zandi-Nejad, K., et al. (2009) Slc26a9 is inhibited by the R-region of CFTR via the STAS domain. J. Biol. Chem. 284, 28306–28318.

    Article  PubMed  CAS  Google Scholar 

  8. Wang, Y., Soyombo, A. A., Shcheynikov, N., Zeng, W., Dorwart, M., Marino, C. R., et al. (2006) Slc26a6 regulates CFTR activity in vivo to determine pancreatic duct HCO(3)(–) secretion: relevance to cystic fibrosis. EMBO J. 25, 5049–5057.

    Article  PubMed  CAS  Google Scholar 

  9. Ko, S. B., Zeng, W., Dorwart, M. R., Luo, X., Kim, K. H., Millen, L., et al. (2004) Gating of CFTR by the STAS domain of SLC26 transporters. Nat. Cell Biol. 6, 343–350.

    Article  PubMed  CAS  Google Scholar 

  10. Seidler, U., Singh, A. K., Cinar, A., Chen, M., Hillesheim, J., Hogema, B., et al. (2009) The role of the NHERF family of PDZ scaffolding proteins in the regulation of salt and water transport. Ann. NY Acad. Sci. 1165, 249–260.

    Article  PubMed  CAS  Google Scholar 

  11. Yang, D., Shcheynikov, N., Zeng, W., Ohana, E., So, I., Ando, H., et al. (2009) IRBIT coordinates epithelial fluid and HCO3-secretion by stimulating the transporters pNBC1 and CFTR in the murine pancreatic duct. J. Clin. Invest. 119, 193–202.

    PubMed  CAS  Google Scholar 

  12. Guggino, W. B., and Stanton, B. A. (2006) New insights into cystic fibrosis: molecular switches that regulate CFTR. Nat. Rev. Mol. Cell Biol. 7, 426–436.

    Article  PubMed  CAS  Google Scholar 

  13. Barasch, J., Kiss, B., Prince, A., Saiman, L., Gruenert, D. C., and Al-Awqati, Q. (1991) Defective acidification of intracellular organelles in cystic fibrosis. Nature 352, 70–73.

    Article  PubMed  CAS  Google Scholar 

  14. Di, A., Brown, M. E., Deriy, L. V., Li, C., Szeto, F. L., Chen, Y., et al. (2006) CFTR regulates phagosome acidification in macrophages and alters bactericidal activity. Nat. Cell Biol. 8, 933–944.

    Article  PubMed  CAS  Google Scholar 

  15. Haggie, P. M., and Verkman, A. S. (2009) Unimpaired lysosomal acidification in respiratory epithelial cells in cystic fibrosis. J. Biol.Chem. 284, 7681–7686.

    Article  PubMed  CAS  Google Scholar 

  16. Jouret, F., Bernard, A., Hermans, C., Dom, G., Terryn, S., Leal, T., et al. (2007) Cystic fibrosis is associated with a defect in apical receptor-mediated endocytosis in mouse and human kidney. J. Am. Soc. Nephrol. 18, 707–718.

    Article  PubMed  CAS  Google Scholar 

  17. Barriere, H., Bagdany, M., Bossard, F., Okiyoneda, T., Wojewodka, G., Gruenert, D., et al. (2009) Revisiting the role of cystic fibrosis transmembrane conductance regulator and counterion permeability in the pH regulation of endocytic organelles. Mol. Biol. Cell 20, 3125–3141.

    Article  PubMed  CAS  Google Scholar 

  18. Kunzelmann, K. (1999) The Cystic Fibrosis Transmembrane Conductance Regulator and its function in epithelial transport. Rev. Physiol. Biochem. Pharmacol. 137, 1–70.

    PubMed  CAS  Google Scholar 

  19. Ribeiro, C. M., Paradiso, A. M., Schwab, U., Perez-Vilar, J., Jones, L., O’Neal, W., et al. (2005) Chronic airway infection/inflammation Induces a Ca2+i-dependent hyperinflammatory response in human cystic fibrosis airway epithelia. J. Biol. Chem. 280, 17798–17806.

    Article  PubMed  CAS  Google Scholar 

  20. Becker, M. N., Sauer, M. S., Muhlebach, M. S., Hirsh, A. J., Wu, Q., Verghese, M. W., et al. (2004) Cytokine secretion by cystic fibrosis airway epithelial cells. Am. J. Respir. Crit. Care Med. 169, 645–653.

    Article  PubMed  Google Scholar 

  21. Hybiske, K., Fu, Z., Schwarzer, C., Tseng, J., Do, J., Huang, N., et al. (2007) Effects of cystic fibrosis transmembrane conductance regulator (CFTR) and delta F508-CFTR on inflammatory response, ER stress and Ca2+ of airway epithelia. Am. J. Physiol. Lung Cell. Mol. Physiol. 293, L1250–L1260.

    Article  PubMed  CAS  Google Scholar 

  22. Perez, A., Issler, A. C., Cotton, C. U., Kelley, T. J., Verkman, A. S., and Davis, P. B. (2007) CFTR inhibition mimics the cystic fibrosis inflammatory profile. Am. J. Physiol. Lung Cell. Mol. Physiol. 292, L383–L395.

    Article  PubMed  CAS  Google Scholar 

  23. Antigny, F., Norez, C., Becq, F., and Vandebrouck, C. (2008) Calcium homeostasis is abnormal in cystic fibrosis airway epithelial cells but is normalized after rescue of F508del-CFTR. Cell Calcium 43, 175–183.

    Article  PubMed  CAS  Google Scholar 

  24. Rottner, M., Kunzelmann, C., Mergey, M., Freyssinet, J. M., and Martinez, M. C. (2007) Exaggerated apoptosis and NF-κB activation in pancreatic and tracheal cystic fibrosis cells. FASEB J. 21, 2939–2948.

    Article  PubMed  Google Scholar 

  25. Hajj, R., Lesimple, P., Nawrocki-Raby, B., Birembaut, P., Puchelle, E., and Coraux, C. (2007) Human airway surface epithelial regeneration is delayed and abnormal in cystic fibrosis. J. Pathol. 211, 340–350.

    Article  PubMed  CAS  Google Scholar 

  26. Lesimple, P., Liao, J., Robert, R., Gruenert, D. C., and Hanrahan, J. W. (2010) CFTR trafficking modulates the barrier function of airway epithelial cell monolayers. J. Physiol. 588, 1195–1209.

    Article  PubMed  CAS  Google Scholar 

  27. 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. Pflügers Arch. 391, 85–100.

    Article  PubMed  CAS  Google Scholar 

  28. Dalemans, W., Barbry, P., Champigny, G., Jallat, S., Dott, K., Dreyer, D., et al. (1992) Altered chloride ion channel kinetics associated with the deltaF508 cystic fibrosis mutation. Nature 354, 526–528.

    Article  Google Scholar 

  29. 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 

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

    Article  PubMed  CAS  Google Scholar 

  31. Bachhuber, T., König, J., Voelcker, T., Mürle, B., Schreiber, R., and Kunzelmann, K. (2005) Chloride interference with the epithelial Na+ channel ENaC. J. Biol. Chem. 280, 31587–31594.

    Article  PubMed  CAS  Google Scholar 

  32. Jiang, Q., Li, J., Dubroff, R., Ahn, Y. J., Foskett, J. K., Engelhardt, J. F., et al. (2000) Epithelial sodium channels regulate cystic fibrosis transmembrane conductance regulator chloride channels in Xenopus oocytes. J. Biol. Chem. 275, 13266–13274.

    Article  PubMed  CAS  Google Scholar 

  33. Ismailov, I. I., Berdiev, B. K., Shlyonsky, V. G., Fuller, C. M., Prat, A. G., Jovov, B., et al. (1997) Role of actin in regulation of epithelial sodium channels by CFTR. Am. J. Physiol. 272, C1077–C1086.

    PubMed  CAS  Google Scholar 

  34. Kunzelmann, K., Mall, M., Briel, M., Hipper, A., Nitschke, R., Ricken, S., et al. (1997) The cystic fibrosis transmembrane conductance regulator attenuates the endogenous Ca2+ activated Cl– conductance in Xenopus ooyctes. Pflügers Arch. 434, 178–181.

    Article  Google Scholar 

  35. Wagner, C. A., Friedrich, B., Setiawan, I., Lang, F., and Broer, S. (2000) The use of Xenopus laevis oocytes for the functional characterization of heterologously expressed membrane proteins. Cell Physiol. Biochem. 10, 1–12.

    Article  PubMed  CAS  Google Scholar 

  36. Treharne, K. J., Xu, Z., Chen, J.-H., Best, O. G., Cassidy, D., Gruenert, D. C., et al. (2009) Inhibition of protein kinase CK2 closes the CFTR Cl– channel, but has no effect on the cystic fibrosis mutant F508-CFTR. Cell Physiol. Biochem. 24, 347–360.

    Article  PubMed  CAS  Google Scholar 

  37. Boucherot, A., Schreiber, R., and Kunzelmann, K. (2001) Role of CFTR’s PDZ-binding domain, NBF1 and Cl– conductance in inhibition of epithelial Na+ channels in Xenopus oocytes. Biochim. Biophys. Acta 1515, 64–71.

    Article  PubMed  CAS  Google Scholar 

  38. Kunzelmann, K., and Nitschke, R. (2000) Defects in processing and trafficking of CFTR. Exp. Nephr. 8, 332–342.

    Article  CAS  Google Scholar 

  39. Cheng, S. H., Rich, D. P., Marshall, J., Gregory, R. J., Welsh, M. J., and Smith, A. E. (1991) Phosphorylation of the R domain by cAMP-dependent protein kinase regulates the CFTR chloride channel. Cell 66, 1027–1036.

    Article  PubMed  CAS  Google Scholar 

  40. Chappe, V., Hinkson, D. A., Zhu, T., Chang, X. B., Riordan, J. R., and Hanrahan, J. W. (2003) Phosphorylation of protein kinase C sites in NBD1 and the R domain control CFTR channel activation by PKA. J. Physiol. 548, 39–52.

    Article  PubMed  CAS  Google Scholar 

  41. Hallows, K. R., Raghuram, V., Kemp, B. E., Witters, L. A., and Foskett, J. K. (2000) Inhibition of cystic fibrosis transmembrane conductance regulator by novel interaction with the metabolic sensor AMP-activated protein kinase. J. Clin. Invest. 105, 1711–1721.

    Article  PubMed  CAS  Google Scholar 

  42. Chang, X.-B., Tabcharani, J. A., Hou, Y.-X., Jensen, T. J., Kartner, N., Alon, N., et al. (1993) Protein kinase A (PKA) still activates CFTR chloride channels after mutagenesis of all 10 PKA consensus phosphorylation sites. J. Biol. Chem. 268, 11304–11311.

    PubMed  CAS  Google Scholar 

  43. Kongsuphol, P., Cassidy, D., Hieke, B., Treharne, K. J., Schreiber, R., Mehta, A., et al. (2009) Mechanistic insight into control of CFTR by AMPK. J. Biol. Chem. 284, 5645–5653.

    Article  PubMed  CAS  Google Scholar 

  44. Kongsuphol, P., Hieke, B., Ousingsawat, J., Almaça, J., Viollet, B., Schreiber, R., et al. (2008) Regulation of Cl– secretion by AMPK in vivo. Pflügers Arch. 457, 1071–1078.

    Article  PubMed  Google Scholar 

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

    PubMed  CAS  Google Scholar 

  46. King, J. D., Jr., Fitch, A. C., Lee, J. K., McCane, J. E., Mak, D. O., Foskett, J. K., et al. (2009) AMP-activated protein kinase phosphorylation of the R domain inhibits PKA stimulation of CFTR. Am. J. Physiol. Cell Physiol. 297, C94–C101.

    Article  PubMed  CAS  Google Scholar 

  47. Koefoed-Johnsen, V., and Ussing, H. H. (1958) The nature of frog skin potential. Acta Physiol. Scand. 42, 298–308.

    Article  PubMed  CAS  Google Scholar 

  48. Li, H., Sheppard, D. N., and Hug, M. J. (2004) Transepithelial electrical measurements with the Ussing chamber. J. Cyst. Fibros. 3(Suppl 2), 123–126.

    Article  PubMed  CAS  Google Scholar 

  49. Mall, M., Hirtz, S., Gonska, T., and Kunzelmann, K. (2004) Assessment of CFTR function in rectal biopsies for the diagnosis of cystic fibrosis. J. Cyst. Fibros. 3, 165–169.

    Article  PubMed  CAS  Google Scholar 

  50. Hirtz, S., Gonska, T., Seydewitz, H. H., Thomas, J., Greiner, P., Kuehr, J., et al. (2004) CFTR Cl– channel function in native human colon correlates with the genotype and the phenotype in cystic fibrosis. Gastroenterology 127, 1085–1095.

    Article  PubMed  CAS  Google Scholar 

  51. Robison, T. W., Dorio, R. J., and Kim, K. J. (1993) Formation of tight monolayers of guinea pig airway epithelial cells cultured in an air-interface: bioelectric properties. Biotechniques 15, 468–473.

    PubMed  CAS  Google Scholar 

  52. Johnson, L. G., Dickman, K. G., Moore, K. L., Mandel, L. J., and Boucher, R. C. (1993) Enhanced Na+ transport in an air-liquid interface culture system. Am. J. Physiol. 264, L560–L565.

    PubMed  CAS  Google Scholar 

  53. Tarran, R., Trout, L., Donaldson, S. H., and Boucher, R. C. (2006) Soluble mediators, not cilia, determine airway surface liquid volume in normal and cystic fibrosis superficial airway epithelia. J. Gen. Physiol. 127, 591–604.

    Article  PubMed  CAS  Google Scholar 

  54. Garcia-Caballero, A., Rasmussen, J. E., Gaillard, E., Watson, M. J., Olsen, J. C., Donaldson, S. H., et al. (2009) SPLUNC1 regulates airway surface liquid volume by protecting ENaC from proteolytic cleavage. Proc. Natl. Acad. Sci. USA 106, 11412–11417.

    Article  PubMed  CAS  Google Scholar 

  55. Kleyman, T. R., Carattino, M. D., and Hughey, R. P. (2009) ENaC at the cutting edge: regulation of epithelial sodium channels by proteases. J. Biol. Chem. 284, 20447–20451.

    Article  PubMed  CAS  Google Scholar 

  56. Kunzelmann, K., and Mall, M. (2003) Pharmacotherapy of the ion transport defect in cystic fibrosis: role of purinergic receptor agonists and other potential therapeutics. Am. J. Resp. Med. 2, 299–309.

    CAS  Google Scholar 

  57. Ousingsawat, J., Martins, J. R., Schreiber, R., Rock, J. R., Harfe, B. D., and Kunzelmann, K. (2009) Loss of TMEM16A causes a defect in epithelial Ca2+ dependent chloride transport. J. Biol. Chem. 284, 28698–28703.

    Article  PubMed  CAS  Google Scholar 

  58. Schreiber, R., Castrop, H., and Kunzelmann, K. (2008) Allergen induced airway hyperresponsiveness is absent in ecto-5′-nucleotidase (CD73) deficient mice. Pflugers Arch. 457, 431–440.

    Article  PubMed  CAS  Google Scholar 

  59. Orlando, R. C., Powell, D. W., Croom, R. D., Berschneider, H. M., Boucher, R. C., and Knowles, M. R. (1989) Colonic and esophageal transepithelial potential difference in cystic fibrosis. Gastroenterology 96, 1041–1048.

    PubMed  CAS  Google Scholar 

  60. Wilschanski, M., Famini, H., Strauss-Liviatan, N., Rivlin, J., Blau, H., Bibi, H., et al. (2001) Nasal potential difference measurements in patients with atypical cystic fibrosis. Eur. Respir. J. 17, 1208–1215.

    Article  PubMed  CAS  Google Scholar 

  61. Singh, A. K., Schultz, B. D., Van Driessche, W., and Bridges, R. J. (2004) Transepithelial fluctuation analysis of chloride secretion. J. Cyst. Fibros. 3(Suppl 2), 127–132.

    Article  PubMed  CAS  Google Scholar 

  62. Galietta, L. J., Jayaraman, S., and Verkman, A. S. (2001) Cell-based assay for high-throughput quantitative screening of CFTR chloride transport agonists. Am. J. Physiol. Cell Physiol. 281, C1734–C1742.

    PubMed  CAS  Google Scholar 

  63. Galietta, L. J., Springsteel, M. F., Eda, M., Niedzinski, E. J., By, K., Haddadin, M. J., et al. (2001) Novel CFTR chloride channel activators identified by screening of combinatorial libraries based on flavone and benzoquinolizinium lead compounds. J. Biol. Chem. 276, 19723–19728.

    Article  PubMed  CAS  Google Scholar 

  64. Miret, J. J., Zhang, J., Min, H., Lewis, K., Roth, M., Charlton, M., et al. (2005) Multiplexed G-protein-coupled receptor Ca2+ flux assays for high-throughput screening. J. Biomol. Screen. 10, 780–787.

    Article  PubMed  CAS  Google Scholar 

  65. Thiagarajah, J. R., Broadbent, T., Hsieh, E., and Verkman, A. S. (2004) Prevention of toxin-induced intestinal ion and fluid secretion by a small-molecule CFTR inhibitor. Gastroenterology 126, 511–519.

    Article  PubMed  CAS  Google Scholar 

  66. Pedemonte, N., Lukacs, G. L., Du, K., Caci, E., Zegarra-Moran, O., Galietta, L. J., et al. (2005) Small-molecule correctors of defective DeltaF508-CFTR cellular processing identified by high-throughput screening. J. Clin. Invest. 115, 2564–2571.

    Article  PubMed  CAS  Google Scholar 

  67. de la Fuente, R., Namkung, W., Mills, A., and Verkman, A. S. (2007) Small molecule screen identifies inhibitors of a human intestinal calcium activated chloride channel. Mol. Pharmacol. 73, 758–768.

    Article  Google Scholar 

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Authors and Affiliations

  1. Department of Physiology, University of Regensburg, 93053, Regensburg, Germany

    Karl Kunzelmann

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  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

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Kunzelmann, K. (2011). Introduction to Section V: Assessment of CFTR Function. 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_26

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  • DOI: https://doi.org/10.1007/978-1-61779-117-8_26

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