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Plasma membrane–localized TMEM16 proteins are indispensable for expression of CFTR

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is the secretory chloride channel in epithelial tissues that has a central role in cystic fibrosis (CF) lung and gastrointestinal disease. A recent publication demonstrates a close association between CFTR and TMEM16A, the calcium-activated chloride channel. Thus, no CFTR chloride currents could be detected in airways and large intestine from mice lacking epithelial expression of TMEM16A. Here, we demonstrate that another plasma membrane–localized TMEM16 paralogue, TMEM16F, can compensate for the lack of TMEM16A. Using TMEM16 knockout mice, human lymphocytes, and a number of human cell lines with endogenous protein expression or heterologous expression, we demonstrate that CFTR can only function in the presence of either TMEM16A or TMEM16F. Double knockout of intestinal epithelial TMEM16A/F expression did not produce offsprings, suggesting a lethal phenotype in utero. Plasma membrane–localized TMEM16A or TMEM16F is required for exocytosis and expression of CFTR in the plasma membrane. TMEM16A/F proteins may therefore have an impact on disease severity in CF.

Key messages

• Cystic fibrosis is caused by the defective Cl channel cystic fibrosis transmembrane conductance regulator (CFTR).

• A close relationship exists between CFTR and the calcium-activated chloride channels TMEM16A/TMEM16F.

• In conditional airway and intestinal knockout mice, lymphocytes from Scott disease patients and in overexpressing cells, CFTR is not functional in the absence of TMEM16A and TMEM16F.

• TMEM16A and TMEM16F support membrane exocytosis and are essential for plasma membrane insertion of CFTR.

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Author information

Correspondence to Karl Kunzelmann.

Ethics declarations

All animal experiments were approved by the local ethics committee of the Government of Unterfranken/Würzburg (AZ: 55.2-2532-2-328) and were conducted according to the guidelines of the American Physiological Society and the German law for the welfare of animals.

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The authors declare that they have no conflict of interest.

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Electronic supplementary material

TMEM16A augments Ca2+induced exocytosis. Video 1: Extracellular application of the styryl lipid dye FM4-64 labels plasma membrane (PM) lipid in mock-transfected HEK293 cells. Additional application of the Ca2+ ionophore ionomycin (1 μM) further augments PM-labeling due to activation of membrane exocytosis. (WMV 207 kb)

Supplementary Figure 1

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Supplementary Figure 2

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Supplementary Figure 3

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Supplementary Figure 4

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Supplementary video 1.

TMEM16A augments Ca2+induced exocytosis. Video 1: Extracellular application of the styryl lipid dye FM4-64 labels plasma membrane (PM) lipid in mock-transfected HEK293 cells. Additional application of the Ca2+ ionophore ionomycin (1 μM) further augments PM-labeling due to activation of membrane exocytosis. (WMV 207 kb)

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Cite this article

Benedetto, R., Ousingsawat, J., Cabrita, I. et al. Plasma membrane–localized TMEM16 proteins are indispensable for expression of CFTR. J Mol Med 97, 711–722 (2019) doi:10.1007/s00109-019-01770-4

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Keywords

  • CFTR
  • Cystic fibrosis transmembrane conductance regulator
  • Cystic fibrosis
  • CF
  • TMEM16A
  • Anoctamin 1
  • TMEM16F
  • Anoctamin 6
  • Ca2+-activated Cl channel
  • Phospholipid scramblase