In situ measurement of airway surface liquid [K+] using a ratioable K+-sensitive fluorescent dye

Wan Namkung, Yuanlin Song, Aaron D. Mills, Prashant Padmawar, Walter E. Finkbeiner, Alan S. Verkman

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

The airway surface liquid (ASL) is the thin fluid layer lining airway surface epithelial cells, whose volume and composition are tightly regulated and may be abnormal in cystic fibrosis (CF). We synthesized a two-color fluorescent dextran to measure ASL [K+], TAC-Lime-dextran-TMR, consisting of a green-fluorescing triazacryptand K+ ionophore-Bodipy conjugate, coupled to dextran, together with a red fluorescing tetramethylrhodamine reference chromophore. TAC-Lime-dextran-TMR fluorescence was K+-selective, increasing >4-fold with increasing [K+] from 0 to 40 mM. In well differentiated human airway epithelial cells, ASL [K+] was 20.8 ± 0.3 mM and decreased by inhibition of the Na+/K+ pump (ouabain), ENaC (amiloride), CF transmembrane conductance regulator (CFTRinh-172), or K+ channels (TEA or XE991). ASL [K+] was increased by forskolin but not affected by Na+/K+/2Cl-cotransporter inhibition (bumetanide). Functional and expression studies indicated the involvement of [K+] channels KCNQ1, KCNQ3, and KCNQ5 as determinants of ASL [K+]. [K+] in CF cultures was similar to that in non-CF cultures, suggesting that abnormal ASL [K+] is not a factor in CF lung disease. In intact airways, ASL [K+] was also well above extracellular [K+]: 22 ± 1 mM in pig trachea ex vivo and 16 ± 1 mM in mouse trachea in vivo. Our results provide the first noninvasive measurements of [K+] in the ASL and indicate the involvement of apical and basolateral membrane ion transporters in maintaining a high ASL [K+].

Original languageEnglish
Pages (from-to)15916-15926
Number of pages11
JournalJournal of Biological Chemistry
Volume284
Issue number23
DOIs
Publication statusPublished - 2009 Jun 5

Fingerprint

Dextrans
Fluorescent Dyes
Cystic Fibrosis
Liquids
Trachea
Epithelial Cells
Bumetanide
Cystic Fibrosis Transmembrane Conductance Regulator
Membrane Transport Proteins
Amiloride
Ionophores
Colforsin
Ouabain
Cell Size
Lung Diseases
Fibrosis
Swine
Color
Fluorescence
Ions

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Namkung, Wan ; Song, Yuanlin ; Mills, Aaron D. ; Padmawar, Prashant ; Finkbeiner, Walter E. ; Verkman, Alan S. / In situ measurement of airway surface liquid [K+] using a ratioable K+-sensitive fluorescent dye. In: Journal of Biological Chemistry. 2009 ; Vol. 284, No. 23. pp. 15916-15926.
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abstract = "The airway surface liquid (ASL) is the thin fluid layer lining airway surface epithelial cells, whose volume and composition are tightly regulated and may be abnormal in cystic fibrosis (CF). We synthesized a two-color fluorescent dextran to measure ASL [K+], TAC-Lime-dextran-TMR, consisting of a green-fluorescing triazacryptand K+ ionophore-Bodipy conjugate, coupled to dextran, together with a red fluorescing tetramethylrhodamine reference chromophore. TAC-Lime-dextran-TMR fluorescence was K+-selective, increasing >4-fold with increasing [K+] from 0 to 40 mM. In well differentiated human airway epithelial cells, ASL [K+] was 20.8 ± 0.3 mM and decreased by inhibition of the Na+/K+ pump (ouabain), ENaC (amiloride), CF transmembrane conductance regulator (CFTRinh-172), or K+ channels (TEA or XE991). ASL [K+] was increased by forskolin but not affected by Na+/K+/2Cl-cotransporter inhibition (bumetanide). Functional and expression studies indicated the involvement of [K+] channels KCNQ1, KCNQ3, and KCNQ5 as determinants of ASL [K+]. [K+] in CF cultures was similar to that in non-CF cultures, suggesting that abnormal ASL [K+] is not a factor in CF lung disease. In intact airways, ASL [K+] was also well above extracellular [K+]: 22 ± 1 mM in pig trachea ex vivo and 16 ± 1 mM in mouse trachea in vivo. Our results provide the first noninvasive measurements of [K+] in the ASL and indicate the involvement of apical and basolateral membrane ion transporters in maintaining a high ASL [K+].",
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In situ measurement of airway surface liquid [K+] using a ratioable K+-sensitive fluorescent dye. / Namkung, Wan; Song, Yuanlin; Mills, Aaron D.; Padmawar, Prashant; Finkbeiner, Walter E.; Verkman, Alan S.

In: Journal of Biological Chemistry, Vol. 284, No. 23, 05.06.2009, p. 15916-15926.

Research output: Contribution to journalArticle

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AU - Song, Yuanlin

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