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.
• 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, log in to check access.
Buy single article
Instant unlimited access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
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
Pedemonte N, Galietta LJ (2014) Structure and function of TMEM16 proteins (anoctamins). Physiol Rev 94:419–459
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
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
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
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
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
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
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
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
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
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
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
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
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
Betz WJ, Mao F, Smith CB (1996) Imaging exocytosis and endocytosis. Curr Opin Neurobiol 6:365–371
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
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
Schuber F, Hong K, Duzgunes N, Papahadjopoulos D (1983) Polyamines as modulators of membrane fusion: aggregation and fusion of liposomes. Biochemistry 22:6134–6140
Rock JR, Harfe BD (2008) Expression of TMEM16 paralogs during murine embryogenesis. Dev Dyn 237:2566–2574
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
Suzuki J, Umeda M, Sims PJ, Nagata S (2010) Calcium-dependent phospholipid scrambling by TMEM16F. Nature 468:834–838
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
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
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
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
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
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
Ammar MR, Kassas N, Chasserot-Golaz S, Bader MF, Vitale N (2013) Lipids in regulated exocytosis: what are they doing? Front Endocrinol 4:125
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
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
Chernomordik LV, Kozlov MM (2005) Membrane hemifusion: crossing a chasm in two leaps. Cell 123:375–382
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
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
Amaral MD, Kunzelmann K (2007) Molecular targeting of CFTR as a therapeutic approach to cystic fibrosis. Trends Pharmacol Sci 28:334–341
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)
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
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
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.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
(PDF 682 kb)
(PDF 2492 kb)
(PDF 391 kb)
(PDF 161 kb)
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)
About this article
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
- Cystic fibrosis transmembrane conductance regulator
- Cystic fibrosis
- Anoctamin 1
- Anoctamin 6
- Ca2+-activated Cl− channel
- Phospholipid scramblase