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How to Measure CFTR-Dependent Bicarbonate Transport: From Single Channels to the Intact Epithelium

  • Martin J. Hug
  • Lane L. Clarke
  • Michael A. GrayEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 741)

Abstract

Bicarbonate serves many functions in our body. It is the predominant buffer maintaining a physiological pH in the blood and within our cells. It is also essential for proper digestion of nutrients and solubilization of complex protein mixtures, such as digestive enzymes and mucins, in epithelial secretions. Transepithelial HCO3 transport also drives net fluid secretion in many epithelial tissues including those in the gastrointestinal and reproductive tracts as well as the airways. Indeed, defective bicarbonate secretion is a hallmark of the pathophysiology in the pancreas of most patients suffering from cystic fibrosis. Some, but not all, disease-causing mutations in the CF gene lead to impaired bicarbonate transport when expressed in heterologous systems. Recently developed pharmacological modulators of mutant CFTR have demonstrated an ability to activate chloride transport but little is known about whether they also increase the secretion of bicarbonate. It is therefore essential to assay bicarbonate transport when studying the effect of small molecules on CFTR function. However, due to the chaotropic nature of the ion, the measurement of the absolute bicarbonate concentration and its permeability through CFTR is far from trivial. In this chapter we will review some of the techniques available to measure bicarbonate transport through single ion channels, individual cells, and intact epithelial layers.

Key words

Bicarbonate transport epithelial cells cystic fibrosis CFTR patch clamp technique pH stat Ussing chambers intracellular pH fluorescent dyes 

Notes

Acknowledgements

MJH acknowledges generous support from Mukoviszidose e.V. (N03/07), Innovative Medizinische Förderung Münster (HU 1 1 01 03), and EuroCare CF (LSHM-CT-2005-018932). LLC acknowledges the technical assistance of Erin E. Hoover and support from the National Institutes of Health (DK48816). MAG gratefully acknowledges support from the Welcome Trust (079673), the Cystic Fibrosis Trust (PJ540), and the Royal Society (2006R1/JP).

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Martin J. Hug
    • 1
  • Lane L. Clarke
    • 2
  • Michael A. Gray
    • 3
    Email author
  1. 1.PharmacyUniversity Medical Center FreiburgFreiburgGermany
  2. 2.Department of Biomedical SciencesDalton Cardiovascular Research Center, University of MissouriColumbiaUSA
  3. 3.Epithelial Research GroupInstitute for Cell and Molecular Biosciences, Newcastle UniversityNewcastle Upon TyneUK

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