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.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

References

  1. 1.

    Greger R, Schreiber R, Mall M, Wissner A, Hopf A, Briel M, Bleich M, Warth R, Kunzelmann K (2001) Cystic fibrosis and CFTR. Pflugers Arch 443:S3–S7

    Article  CAS  PubMed  Google Scholar 

  2. 2.

    Pedemonte N, Galietta LJ (2014) Structure and function of TMEM16 proteins (anoctamins). Physiol Rev 94:419–459

    Article  CAS  PubMed  Google Scholar 

  3. 3.

    Schreiber R, Faria D, Skryabin BV, Rock JR, Kunzelmann K (2014) Anoctamins support calcium-dependent chloride secretion by facilitating calcium signaling in adult mouse intestine. Pflugers Arch 467:1203–1213

    Article  CAS  PubMed  Google Scholar 

  4. 4.

    Ousingsawat J, Kongsuphol P, Schreiber R, Kunzelmann K (2011) CFTR and TMEM16A are separate but functionally related Cl channels. Cell Physiol Biochem 28:715–724

    Article  CAS  PubMed  Google Scholar 

  5. 5.

    Benedetto R, Ousingsawat J, Wanitchakool P, Zhang Y, Holtzman MJ, Amaral M, Rock JR, Schreiber R, Kunzelmann K (2017) Epithelial chloride transport by CFTR requires TMEM16A. Sci Rep 7:12397

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Kunzelmann K, Kongsuphol P, AlDehni F, Tian Y, Ousingsawat J, Warth R, Schreiber R (2009) Bestrophin and TMEM16 - Ca2+ activated Cl- channels with different functions. Cell Calcium 46:233–241

    Article  CAS  PubMed  Google Scholar 

  7. 7.

    Schreiber R, Uliyakina I, Kongsuphol P, Warth R, Mirza M, Martins JR, Kunzelmann K (2010) Expression and function of epithelial anoctamins. J Biol Chem 285:7838–7845

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Sirianant L, Wanitchakool P, Ousingsawat J, Benedetto R, Zormpa A, Cabrita I, Schreiber R, Kunzelmann K (2016) Non-essential contribution of LRRC8A to volume regulation. Pflugers Arch 468:1789–1796

    Google Scholar 

  9. 9.

    Kmit A, van Kruchten R, Ousingsawat J, Mattheij NJ, Senden-Gijsbers B, Heemskerk JW, Bevers EM, Kunzelmann K (2013) Calcium-activated and apoptotic phospholipid scrambling induced by Ano6 can occur independently of Ano6 ion currents. Cell Death Dis 25(4):e611

    Article  CAS  Google Scholar 

  10. 10.

    Botelho HM, Uliyakina I, Awatade NT, Proenca MC, Tischer C, Sirianant L, Kunzelmann K, Pepperkok R, Amaral MD (2015) Protein traffic disorders: an effective high-throughput fluorescence microscopy pipeline for drug discovery. Sci Rep 5:9038

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Schenk LK, Ousingsawat J, Skryabin BV, Schreiber R, Pavenstadt H, Kunzelmann K (2018) Regulation and function of TMEM16F in renal podocytes. Int J Mol Sci 19. DOI https://doi.org/10.3390/ijms19061798

  12. 12.

    Son M, Ito Y, Sato S, Ishikawa T, Kondo M, Nakayama S, Shimokata K, Kume H (2004) Apical and basolateral ATP-induced anion secretion in polarized human airway epithelia. Am J Respir Cell Mol Biol 30:411–419

    Article  CAS  PubMed  Google Scholar 

  13. 13.

    Benedetto R, Cabrita I, Schreiber R, Kunzelmann K (2019) TMEM16A is indispensable for basal mucus secretion in airways and intestine. FASEB J (in press) 19. https://doi.org/10.3390/ijms19061798

  14. 14.

    Lerias J, Pinto M, Benedetto R, Schreiber R, Amaral M, Aureli M, Kunzelmann K (2018) Compartmentalized crosstalk of CFTR and TMEM16A (ANO1) through EPAC1 and ADCY1. Cell Signal 44:10–19

    Article  CAS  PubMed  Google Scholar 

  15. 15.

    Bricogne C, Fine M, Pereira PM, Sung J, Tijani M, Wang Y, Henriques R, Collins MK, Hilgemann D (2019) TMEM16F activation by Ca(2+) triggers plasma membrane expansion and directs PD-1 trafficking. Sci Rep 9:619

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Betz WJ, Mao F, Smith CB (1996) Imaging exocytosis and endocytosis. Curr Opin Neurobiol 6:365–371

    Article  CAS  PubMed  Google Scholar 

  17. 17.

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

    Article  CAS  Google Scholar 

  18. 18.

    Schiavo G, Benfenati F, Poulain B, Rossetto O, Polverino de Laureto P, DasGuptal BR, Montecucco C (1992) Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin. Nature 359:832–825

    Article  CAS  PubMed  Google Scholar 

  19. 19.

    Schuber F, Hong K, Duzgunes N, Papahadjopoulos D (1983) Polyamines as modulators of membrane fusion: aggregation and fusion of liposomes. Biochemistry 22:6134–6140

    Article  CAS  PubMed  Google Scholar 

  20. 20.

    Rock JR, Harfe BD (2008) Expression of TMEM16 paralogs during murine embryogenesis. Dev Dyn 237:2566–2574

    Article  CAS  PubMed  Google Scholar 

  21. 21.

    Mattheij NJ, Braun A, van Kruchten R, Castoldi E, Pircher J, Baaten CC, Wulling M, Kuijpers MJ, Kohler R, Poole AW et al (2015) Survival protein anoctamin-6 controls multiple platelet responses including phospholipid scrambling, swelling, and protein cleavage. FASEB J 30:727–737

    Article  CAS  PubMed  Google Scholar 

  22. 22.

    Suzuki J, Umeda M, Sims PJ, Nagata S (2010) Calcium-dependent phospholipid scrambling by TMEM16F. Nature 468:834–838

    Article  CAS  PubMed  Google Scholar 

  23. 23.

    Jin X, Shah S, Liu Y, Zhang H, Lees M, Fu Z, Lippiat JD, Beech DJ, Sivaprasadarao A, Baldwin SA et al (2013) Activation of the Cl- channel ANO1 by localized calcium signals in nociceptive sensory neurons requires coupling with the IP3 receptor. Sci Signal 6:ra73

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Cabrita I, Benedetto R, Fonseca A, Wanitchakool P, Sirianant L, Skryabin BV, Schenk LK, Pavenstadt H, Schreiber R, Kunzelmann K (2017) Differential effects of anoctamins on intracellular calcium signals. FASEB J 31:2123–2134

    Article  CAS  PubMed  Google Scholar 

  25. 25.

    Ousingsawat J, Wanitchakool P, Kmit A, Romao AM, Jantarajit W, Schreiber S, Kunzelmann K (2015) Anoctamin 6 mediates effects essential for innate immunity downstream of P2X7-receptors in macrophages. Nat Commun 6:6245

    Article  CAS  PubMed  Google Scholar 

  26. 26.

    Ousingsawat J, Wanitchakool P, Schreiber R, Wuelling M, Vortkamp A, Kunzelmann K (2015) Anoctamin 6 controls bone mineralization by activating the calcium transporter NCX1. J Biol Chem 290:6270–6280

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Ousingsawat J, Cabrita I, Wanitchakool P, Sirianant L, Krautwald S, Linkermann A, Schreiber R, Kunzelmann K (2016) Ca2+ signals, cell membrane disintegration, and activation of TMEM16F during necroptosis. Cell Mol Life Sci 74:173–181

    Article  CAS  PubMed  Google Scholar 

  28. 28.

    Kunzelmann K, Tian Y, Martins JR, Faria D, Kongsuphol P, Ousingsawat J, Thevenod F, Roussa E, Rock JR, Schreiber R (2011) Anoctamins. Pflugers Arch 462:195–208

    Article  CAS  PubMed  Google Scholar 

  29. 29.

    Ammar MR, Kassas N, Chasserot-Golaz S, Bader MF, Vitale N (2013) Lipids in regulated exocytosis: what are they doing? Front Endocrinol 4:125

    Article  Google Scholar 

  30. 30.

    Ory S, Ceridono M, Momboisse F, Houy S, Chasserot-Golaz S, Heintz D, Calco V, Haeberle AM, Espinoza FA, Sims PJ, Bailly Y, Bader MF, Gasman S (2013) Phospholipid scramblase-1-induced lipid reorganization regulates compensatory endocytosis in neuroendocrine cells. J Neurosci 33:3545–3556

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Yeung T, Gilbert GE, Shi J, Silvius J, Kapus A, Grinstein S (2008) Membrane phosphatidylserine regulates surface charge and protein localization. Science 319:210–213

    Article  CAS  PubMed  Google Scholar 

  32. 32.

    Chernomordik LV, Kozlov MM (2005) Membrane hemifusion: crossing a chasm in two leaps. Cell 123:375–382

    Article  CAS  PubMed  Google Scholar 

  33. 33.

    He M, Ye W, Wang WJ, Sison ES, Jan YN, Jan LY (2017) Cytoplasmic Cl(-) couples membrane remodeling to epithelial morphogenesis. Proc Natl Acad Sci U S A 114:E11161–e11169

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Henkel B, Drose DR, Ackels T, Oberland S, Spehr M, Neuhaus EM (2015) Co-expression of anoctamins in cilia of olfactory sensory neurons. Chem Senses 40:73–87

    Article  CAS  PubMed  Google Scholar 

  35. 35.

    Amaral MD, Kunzelmann K (2007) Molecular targeting of CFTR as a therapeutic approach to cystic fibrosis. Trends Pharmacol Sci 28:334–341

    Article  CAS  PubMed  Google Scholar 

  36. 36.

    Kunzelmann K, Ousingsawat J, Cabrita I, Doušová T, Bähr A, Janda M, Schreiber R, Benedetto R (2019) TMEM16A in cystic fibrosis: activating or inhibiting? Front Pharmacol (in press)

  37. 37.

    Wang P, Zhao W, Sun J, Tao T, Chen X, Zheng YY, Zhang CH, Chen Z, Gao YQ, She F et al (2018) Inflammatory mediators mediate airway smooth muscle contraction through a G protein-coupled receptor-transmembrane protein 16A-voltage-dependent Ca(2+) channel axis and contribute to bronchial hyperresponsiveness in asthma. J Allergy Clin Immunol 141:1259–1268.e1211

    Article  CAS  PubMed  Google Scholar 

  38. 38.

    Miner K, Labitzke K, Liu B, Elliot R, Wang P, Henckels K, Gaida K, Elliot R, Chen JJ, Liu L et al (2019) The anthelminthic niclosamide and related compounds represent potent Tmem16a antagonists that fully relax mouse and human airway rings. Froniers in Pharmacology 35:411–413

    Google Scholar 

Download references

Funding

This study is supported by UK CF Trust SRC013, DFG KU756/14-1, DFG Projektnummer 387509280—SFB 1350, and Gilead Stiftung.

Author information

Affiliations

Authors

Corresponding author

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.

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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

(PDF 682 kb)

Supplementary Figure 2

(PDF 2492 kb)

Supplementary Figure 3

(PDF 391 kb)

Supplementary Figure 4

(PDF 161 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

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). https://doi.org/10.1007/s00109-019-01770-4

Download citation

Keywords

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